Methods for treating hypercholesterolemia using antibodies to pcsk9

ABSTRACT

The present invention provides methods for treating hypercholesterolemia. The methods of the present invention comprise administering to a subject in need thereof a therapeutic composition comprising an anti-PCSK9 antibody or antigen-binding fragment thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.13/095,234, filed on Apr. 27, 2011, which is a continuation-in-part ofU.S. application Ser. No. 12/637,942, filed on Dec. 15, 2009, now U.S.Pat. No. 8,062,640, which claims the benefit under 35 USC §119(e) ofU.S. Provisional Patent Appl. Nos. 61/122,482, filed on Dec. 15, 2008;61/210,566, filed on Mar. 18, 2009; 61/168,753, filed on Apr. 13, 2009;61/218,136, filed on Jun. 18, 2009; 61/249,135, filed on Oct. 6, 2009;and 61/261,776, filed on Nov. 17, 2009, which applications are hereinspecifically incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention is related to human antibodies and antigen-bindingfragments of human antibodies that specifically bind human proproteinconvertase subtilisin/kexin type 9 (PCSK9), and therapeutic methods ofusing those antibodies.

STATEMENT OF RELATED ART

Proprotein convertase subtilisin/kexin, type 9 (PCSK9) is a proproteinconvertase belonging to the proteinase K subfamily of the secretorysubtilase family. The encoded protein is synthesized as a solublezymogen that undergoes autocatalytic intramolecular processing in theendoplasmic reticulum. Evidence suggest that PCSK9 increases plasma LDLcholesterol by promoting degradation of the LDL receptor, which mediatesLDL endocytosis in the liver, the major route of LDL clearance fromcirculation. The structure of PCSK9 protein shows that it has a signalsequence, followed by a prodomain, a catalytic domain that contains aconserved triad of residues (D186, H226 and S386), and a C-terminaldomain. It is synthesized as a soluble 74-kDa precursor that undergoesautocatalytic cleavage in the ER, generating a 14-kDa prodomain and60-kDa catalytic fragment. The autocatalytic activity has been shown tobe required for secretion. After cleavage the prodomain remains tightlyassociated with the catalytic domain.

Antibodies to PCSK9 are described in, for example, WO 2008/057457, WO2008/057458, WO 2008/057459, WO 2008/063382, WO 2008/125623, and US2008/0008697.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides fully human monoclonalantibodies (mAbs) and antigen-binding fragments thereof thatspecifically bind and neutralize human PCSK9 (hPCSK9) activity.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum total cholesterol at least about 25-35%and sustaining the reduction over at least a 24 day period relative to apredose level, preferably the reduction in serum total cholesterol is atleast about 30-40%;

(ii) capable of reducing serum LDL cholesterol at least about 65-80% andsustaining the reduction over at least a 24 day period relative to apredose level;

(iii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;

(iv) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose level.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody that specifically binds hPCSK9and is characterized by at least one of:

(i) capable of reducing serum LDL cholesterol at least about 40-70% andsustaining the reduction over at least a 60 or 90 day period relative toa predose level;

(ii) capable of reducing serum triglyceride at least about 25-40%relative to predose level;

(iii) does not reduce serum HDL cholesterol or reduces serum HDLcholesterol no more than 5% relative to predose

In one embodiment, the antibody or antigen-binding fragment ischaracterized as binding an epitope comprising amino acid residue 238 ofhPCSK9 (SEQ ID NO:755). In a more specific embodiment, the antibody orantigen-binding fragment binds an epitope comprising one or more ofamino acid residue 238, 153, 159 and 343 of hPCSK9 (SEQ ID NO:755).). Ina more specific embodiment, the antibody or fragment thereof ischaracterized as binding an epitope which does not comprise an aminoacid residue at position 192, 194, 197 and/or 237 of SEQ ID NO:755.

In one embodiment, the antibody or antigen-binding fragment ischaracterized as binding an epitope comprising amino acid residue 366 ofhPCSK9 (SEQ ID NO:755). In a more specific embodiment, the antibody orantigen-binding fragment binds an epitope comprising one or more ofamino acid residue at position 147, 366 and 380 of SEQ ID NO:755. In amore specific embodiment, the antibody or antigen-binding fragment of anantibody is characterized as binding an epitope which does not comprisean amino acid residue at position 215 or 238 of SEQ ID NO:755.

In one embodiment, the antibody or antigen-binding fragment ischaracterized as exhibiting an enhanced binding affinity (K_(D)) forhPCSK9 at pH 5.5 relative to the K_(D) at pH 7.4, as measured by plasmonsurface resonance. In a specific embodiment, the antibody or fragmentthereof exhibits at least a 20-fold, at least a 40-fold or at least a50-fold enhanced affinity for PCSK9 at an acidic pH relative to aneutral pH, as measured by surface plasmon resonance.

In one embodiment, the antibody or antigen-binding fragment ischaracterized as not exhibiting an enhanced binding affinity for PCSK9at an acidic pH relative to a neutral pH, as measured by surface plasmonresonance. In a specific embodiment, the antibody or fragment thereofexhibits a decreased binding affinity at an acidic pH.

In another embodiment, the antibody or antigen-binding fragment bindshuman, human GOF mutation D374Y, cynomolgus monkey, rhesus monkey,mouse, rat and hamster PCSK9.

In one embodiment, the antibody or antigen-binding fragment binds humanand monkey PCSK9, but does not bind mouse, rat or hamster PCSK9.

The mAbs can be full-length (e.g., an IgG1 or IgG4 antibody) or maycomprise only an antigen-binding portion (e.g., a Fab, F(ab′)₂ or scFvfragment), and may be modified to affect functionality, e.g., toeliminate residual effector functions (Reddy et al. (2000) J. Immunol.164:1925-1933).

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody comprising a heavy chainvariable region (HCVR) selected from the group consisting of SEQ IDNO:2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122,138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238,242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334, 338, 354,358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434, 450, 454, 458,474, 478, 482, 498, 502, 506, 522, 526, 530, 546, 550, 554, 570, 574,578, 594, 598, 602, 618, 622, 626, 642, 646, 650, 666, 670, 674, 690,694, 698, 714, 718, 722, 738 and 742, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity, in one embodiment, the HCVR comprises anamino acid sequence selected from the group consisting of SEQ ID NO:50,66, 70, 74, 90, 94, 122, 138, 142, 218, 234, 238, 242, 258, 262, 314,330 and 334. In a more specific embodiment, the HCVR comprises SEQ IDNO:90 or 218.

In one embodiment, the antibody or fragment thereof further comprises alight chain variable region (LCVR) selected from the group consisting ofSEQ ID NO:10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120,130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236,240, 250, 260, 264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346,356, 360, 370, 380, 384, 394, 404, 408, 418, 428, 432, 442, 452, 456,466, 476, 480, 490, 500, 504, 514, 524, 528, 538, 548, 552, 562, 572,576, 586, 596, 600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682,692, 696, 706, 716, 720, 730, 740 and 744, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity. In one embodiment, the LCVR comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 58,68, 72, 82, 92, 96, 130, 140, 144, 226, 236, 240, 250, 260, 264, 322,332 and 336. In a more specific embodiment, the LCVR comprises SEQ IDNO:92 or 226.

In specific embodiments, the antibody or fragment thereof comprises aHCVR and LCVR (HCVR/LCVR) sequence pair selected from the groupconsisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58,66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130,138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192,194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260,262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322,330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384,386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452,454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514,522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576,578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644,646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696, 698/706,714/716, 718/720, 722/730, 738/740 and 742/744. In one embodiment, theHCVR and LCVR sequence pair comprises one of SEQ ID NO: 50/58, 66/68,70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/226, 234/236,238/240, 242/250, 258/260, 262/264, 314/322, 330/332 and 334/336. In amore specific embodiment, the HCVR/LCVR pair comprises SEQ ID NO:90/92or 218/226.

In a second aspect, the invention features an antibody orantigen-binding fragment of an antibody comprising a heavy chain CDR3(HCDR3) domain selected from the group consisting of SEQ ID NO:8, 32,56, 80, 104, 128, 152, 176, 200, 224, 248, 272, 296, 320, 344, 368, 392,416, 440, 464, 488, 512, 536, 560, 584, 608, 632, 656, 680, 704 and 728,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; and a lightchain CDR3 (LCDR3) domain selected from the group consisting of SEQ IDNO:16, 40, 64, 88, 112, 136, 160, 184, 208, 232, 256, 280, 304, 328,352, 376, 400, 424, 448, 472, 496, 520, 544, 568, 592, 616, 640, 664,688, 712 and 736, or substantially similar sequences thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.In one embodiment, the HCDR3/LCDR3 sequence pair is selected from thegroup consisting of SEQ ID NO:56/64, 80/88, 128/136, 224/232, 248/256and 320/328. In a more specific embodiment, the HCDR3/LCDR3 sequencepair comprises SEQ ID NO:80/88 or 224/232.

In a further embodiment, the invention comprising an antibody orfragment thereof further comprising a heavy chain CDR1 (HCDR1) domainselected from the group consisting of SEQ ID NO:4, 28, 52, 76, 100, 124,148, 172, 196, 220, 244, 268, 292, 316, 340, 364, 388, 412, 436, 460,484, 508, 532, 556, 580, 604, 628, 652, 676, 700 and 724, or asubstantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2(HCDR2) domain selected from the group consisting of SEQ ID NO:6, 30,54, 78, 102, 126, 150, 174, 198, 222, 246, 270, 294, 318, 342, 366, 390,414, 438, 462, 486, 510, 534, 558, 582, 606, 630, 654, 678, 702 and 726,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity, a light chainCDR1 (LCDR1) domain selected from the group consisting of SEQ ID NO:12,36, 60, 84, 108, 132, 156, 180, 204, 228, 252, 276, 300, 324, 348, 372,396, 420, 444, 468, 492, 516, 540, 564, 588, 612, 636, 660, 684, 708 and732, or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity; and a lightchain CDR2 (LCDR2) domain selected from the group consisting of SEQ IDNO:14, 38, 62, 86, 110, 134, 158, 182, 206, 230, 254, 278, 302, 326,350, 374, 398, 422, 446, 470, 494, 518, 542, 566, 598, 614, 638, 662,686, 710 and 734, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.In one embodiment, the heavy and light chain CDR sequences comprise asequence selected from the group consisting of SEQ ID NO:52, 54, 56, 60,62, 64; 76, 78, 80, 84, 86, 88; 124, 126, 128, 132, 134, 136, 220, 222,224, 228, 230, 232; 244, 246, 248, 252, 254, 256; and 316, 318, 320,324, 326, 328. In more specific embodiments, the CDR sequences compriseSEQ ID NO: 76, 78, 80, 84, 86, 88; or 220, 222, 224, 228, 230, 232.

In a related embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody which specifically binds hPCSK9,wherein the antibody or fragment comprises heavy and light chain CDRdomains contained within heavy and light chain sequence pairs selectedfrom the group consisting of SEQ ID NO: 2/10, 18/20, 22/24, 26/34,42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116,118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178,186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240,242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308,310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370,378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432,434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500,502/504, 506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562,570/572, 574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624,626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692,694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744. In oneembodiment, the CDR sequences are contained within HCVR and LCVRselected from the amino acid sequence pairs of SEQ ID NO: 50/58, 66/68,70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/226, 234/236,238/240, 242/250, 258/260, 262/264, 314/322, 330/332 and 334/336. Inmore specific embodiments, the CDR sequences are comprised withinHCVR/LCVR sequences selected from SEQ ID NO: 90/92 or 218/226.

In one embodiment, the invention provides fully human monoclonalantibody or antigen-binding fragment thereof that specifically bindhPCSK9 and neutralize PCSK9 activity, wherein the antibody or fragmentthereof exhibits one or more of the following characteristics: (i)capable of reducing serum total cholesterol at least about 25-35% andsustaining the reduction over at least a 24 day period relative to apredose level, preferably the reduction in serum total cholesterol is atleast about 30-40%; (ii) capable of reducing serum LDL cholesterol atleast about 65-80% and sustaining the reduction over at least a 24 dayperiod relative to a predose level; (iii) capable of reducing serumtriglyceride at least about 25-40% relative to predose level; (iv) doesnot reduce serum HDL cholesterol or reduces serum HDL cholesterol nomore than 5% relative to predose level; (v) binds an epitope comprisingamino acid residue 238 of hPCSK9 (SEQ ID NO:755); (vi) exhibits anenhanced binding affinity (K_(D)) for hPCSK9 at pH 5.5 relative to theK_(D) at pH 7.4, as measured by plasmon surface resonance, wherein theenhanced affinity is at least about a 20- to 50-fold increase inaffinity; (vii) binds human, human GOF mutation D374Y, cynomolgusmonkey, rhesus monkey, mouse, rat and hamster PCSK9; (viii) comprisesheavy and light chain CDR3 sequences comprising SEQ ID NO:80 and 88; and(ix) comprises CDR sequences from SEQ ID NO:90 and 92.

In one embodiment, the invention provides fully human monoclonalantibody or antigen-binding fragment thereof that specifically bindhuman PCSK9 (hPCSK9) and neutralize PCSK9 activity, wherein the antibodyor fragment thereof exhibits one or more of the followingcharacteristics: (i) capable of reducing serum LDL cholesterol at leastabout 40-70% and sustaining the reduction over at least a 60 or 90 dayperiod relative to a predose level; (ii) capable of reducing serumtriglyceride at least about 25-40% relative to predose level; (iii) doesnot reduce serum HDL cholesterol or reduces serum HDL cholesterol nomore than 5% relative to predose level; (iv) binds an epitope comprisingamino acid residue 366 of hPCSK9 (SEQ ID NO:755); (v) does not exhibitan enhanced binding affinity for PCSK9 at an acidic pH relative to aneutral pH, as measured by surface plasmon resonance; (vi) binds humanand monkey PCSK9, but does not bind mouse, rat or hamster PCSK9; (vii)comprises heavy and light chain CDR3 sequences comprising SEQ ID NO:224and 232; and (viii) comprises CDR sequences from SEQ ID NO:218 and 226.

In a third aspect, the invention provides nucleic acid moleculesencoding anti-PCSK9 antibodies or fragments thereof. Recombinantexpression vectors carrying the nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of producing the antibodies byculturing the host cells under conditions permitting production of theantibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 21, 25, 41, 45, 49, 65,69, 73, 89, 93, 97, 113, 117, 121, 137, 141, 145, 161, 165, 169, 185,189, 193, 209, 213, 217, 233, 237, 241, 257, 261, 265, 281, 285, 289,305, 309, 313, 329, 333, 337, 353, 357, 361, 377, 381, 385, 401, 405,409, 425, 429, 433, 449, 453, 457, 473, 477, 481, 497, 501, 505, 521,525, 529, 545, 549, 553, 569, 573, 577, 593, 597, 601, 617, 621, 625,641, 645, 649, 665, 669, 673, 689, 693, 697, 713, 717, 721, 737 and 741,or a substantially identical sequence having at least 90%, at least 95%,at least 98%, or at least 99% homology thereof. In one embodiment, theHCVR is encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 49, 65, 69, 73, 89, 93, 121, 137, 141, 217,233, 237, 241, 257, 261, 313, 329 and 333. In more specific embodiments,the HCVR is encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 89 and 217.

In one embodiment, the antibody or fragment thereof further comprises aLCVR encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 9, 19, 23, 33, 43, 47, 57, 67, 71, 81, 91, 95,105, 115, 119, 129, 139, 143, 153, 163, 167, 177, 187, 191, 201, 211,215, 225, 235, 239, 249, 259, 263, 273, 283, 287, 297, 307, 311, 321,331, 335, 345, 355, 359, 369, 379, 383, 393, 403, 407, 417, 427, 431,441, 451, 455, 465, 475, 479, 489, 499, 503, 513, 523, 527, 537, 547,551, 561, 571, 575, 585, 595, 599, 609, 619, 623, 633, 643, 647, 657,667, 671, 681, 691, 695, 705, 715, 719, 729, 739 and 743, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof. In one embodiment, the LCVRis encoded by a nucleic acid sequence selected from the group consistingof SEQ ID NO: 57, 67, 71, 81, 91, 95, 129, 139, 143, 225, 235, 239, 249,259, 263, 321, 331 and 335. In more specific embodiments, the LCVR isencoded by a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 91 and 225

In one embodiment, the invention features an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO:7,31, 55, 79, 103, 127, 151, 175, 199, 223, 247, 271, 295, 319, 343, 367,391, 415, 439, 463, 487, 511, 535, 559, 583, 607, 631, 655, 679, 703 and727, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; and a LCDR3 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 15, 39, 63, 87, 111, 135, 159, 183, 207, 231, 255, 279, 303,327, 351, 375, 399, 423, 447, 471, 495, 519, 543, 567, 591, 615, 639,663, 687, 711 and 735, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof.In one embodiment, the HCDR3 and LCDR3 comprise a sequence pair encodedby the nucleic acid sequence of SEQ ID NO: 55/63, 79/87, 127/135,223/231, 247/255 and 319/327, respectively. In more specificembodiments, the HCDR3 and LCDR3 comprise a sequence pair encoded by thenucleic acid sequence of SEQ ID NO: 79/87 and 223/231.

In a further embodiment, the antibody or fragment thereof furthercomprises, a HCDR1 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 3, 27, 51, 75, 99, 123, 147, 171,195, 219, 243, 267, 291, 315, 339, 363, 387, 411, 435, 459, 483, 507,531, 555, 579, 603, 627, 651, 675, 699 and 723, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% homology thereof; a HCDR2 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO:5, 29, 53, 77,101, 125, 149, 173, 197, 221, 245, 269, 293, 317, 341, 365, 389, 413,437, 461, 485, 509, 533, 557, 581, 605, 629, 653, 677, 701 and 725, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% homology thereof; a LCDR1 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 11,35, 59, 83, 107, 131, 155, 179, 203, 227, 251, 275, 299, 323, 347, 371,395, 419, 443, 467, 491, 515, 539, 563, 587, 611, 635, 659, 683, 707 and731, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% homology thereof; and a LCDR2 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 13, 37, 61, 85, 109, 133, 157, 181, 205, 229, 253, 277, 301,325, 349, 373, 397, 421, 445, 469, 493, 517, 541, 565, 589, 613, 637,661, 685, 709 and 733, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% homology thereof.In one embodiment, the heavy and light chain CDR sequences are encodedby the nucleic acid sequences of SEQ ID NO: 51, 53, 55, 59, 61, 63; 75,77, 79, 83, 85, 87; 123, 125, 127, 131, 133, 135; 219, 221, 223, 227,229, 231; 243, 245, 247, 251, 253, 255; and 315, 317, 319, 323, 325,327. In more specific embodiments, the heavy and light chain CDRsequences are encoded by the nucleic acid sequences of SEQ ID NO: 75,77, 79, 83, 85, 87; and 219, 221, 223, 227, 229, 231.

In a fourth aspect, the invention features an isolated antibody orantigen-binding fragment thereof that specifically binds hPCSK9,comprising a HCDR3 and a LCDR3, wherein HCDR3 comprises an amino acidsequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶-X¹⁷-X¹⁸-X¹⁹-X²⁰(SEQ ID NO:747), wherein X¹ is Ala, X² is Arg or Lys, X³ is Asp, X⁴ isSer or Ile, X⁵ is Asn or Val, X⁶ is Leu or Trp, X⁷ is Gly or Met, X⁸ isAsn or Val, X⁹ is Phe or Tyr, X¹⁰ is Asp, X¹¹ is Leu or Met, X¹² is Aspor absent, X¹³ is Tyr or absent, X¹⁴ is Tyr or absent, X¹⁵ is Tyr orabsent, X¹⁶ is Tyr or absent, X¹⁷ is Gly or absent, X¹⁸ is Met orabsent, X¹⁹ is Asp or absent, and X²⁰ is Vai or absent; and LCDR3comprises an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:750), wherein X¹ is Gln or Met, X²is Gln, X³ is Tyr or Thr, X⁴ is Tyr or Leu, X⁵ is Thr or Gln, X⁶ is Thr,X⁷ is Pro, X⁸ is Tyr or Leu, and X⁹ is Thr.

In a further embodiment, the antibody or fragment thereof furthercomprise a HCDR1 sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ IDNO:745), wherein X¹ is Gly, X² is Phe, X³ is Thr, X⁴ is Phe, X⁵ is Seror Asn, X⁶ is Ser or Asn, X⁷ is Tyr or His, and X⁸ is Ala or Trp; aHCDR2 sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸ (SEQ ID NO:746),wherein X¹ is Ile, X² is Ser or Asn, X³ is Gly or Gln, X⁴ is Asp or Ser,X⁵ is Gly, X⁶ is Ser or Gly, X⁷ is Thr or Glu, and X⁸ is Thr or Lys; aLCDR1 sequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²(SEQ ID NO:748) wherein X¹ is Gln, X² is Ser, X³ is Val or Leu, X⁴ isLeu, X⁵ is His or Tyr, X⁶ is Arg or Ser, X⁷ is Ser or Asn, X⁸ is Asn orGly, X⁹ is Asn, X¹⁰ is Arg or Asn, X¹¹ is Asn or Tyr, and X¹² is Phe orabsent; a LCDR2 sequence of the formula X¹-X²-X³ (SEQ ID NO:749) whereinX¹ is Trp or Leu, X² is Ala or Gly, and X³ is Ser. FIG. 1 shows thesequence alignment of heavy and light chain variable regions for 316Pand 300N mAbs.

In a fifth aspect, the invention features a human anti-PCSK9 antibody orantigen-binding fragment of an antibody comprising a heavy chainvariable region (HCVR) encoded by nucleotide sequence segments derivedfrom V_(H), D_(H) and J_(H) germline sequences, and a light chainvariable region (LCVR) encoded by nucleotide sequence segments derivedfrom V_(K) and J_(K) germline sequences, wherein the germline sequencesare (a) V_(H) gene segment 3-23, D_(H) gene segment 7-27, J_(H) genesegment 2, V_(K) gene segment 4-1 and J_(K) gene segment 2; or (b) V_(H)gene segment 3-7, D_(H) gene segment 2-8, J_(H) gene segment 6, V_(K)gene segment 2-28 and J_(K) gene segment 4.

In a sixth aspect, the invention features an antibody or antigen-bindingfragment thereof that binds to a PCSK9 protein of SEQ ID NO:755, whereinthe binding of the antibody or fragment thereof to a variant PCSK9protein is less than 50% of the binding between the antibody or fragmentthereof and the PCSK9 protein of SEQ ID NO:755. In specific embodiment,the antibody or fragment thereof binds to the variant PCSK9 protein witha binding affinity (K_(D)) which is less than about 50%, less than about60%, less than about 70%, less than about 80%, less than about 90% orless than about 95% compared to the binding to PCSK9 (SEQ ID NO:755).

In one embodiment, the variant PCSK9 protein comprises at least onemutation at position 238 of SEQ ID NO:755. In a more specificembodiment, the mutation is D238R. In one embodiment, the antibody orantibody fragment binding affinity for the variant PCSK9 protein is atleast 90% less relative to the wildtype protein of SEQ ID NO:755,wherein the variant protein comprises a mutation at residue 238. In oneembodiment, the antibody or antibody fragment binding affinity for thevariant PCSK9 protein is at least 80% less relative to the wildtypeprotein of SEQ ID NO:755, wherein the variant protein comprises amutation at one or more of residue 153, 159, 238 and 343. In a morespecific embodiment, the mutation is one of S153R, E159R, D238R andD343R.

In one embodiment, the variant PCSK9 protein comprises at least onemutation at position 366 of SEQ ID NO:755. In a more specificembodiment, the mutation is E366K. In one embodiment, the antibody orantibody fragment binding affinity for the variant PCSK9 protein is atleast 95% less relative to the wildtype protein of SEQ ID NO:755,wherein the variant protein comprises a mutation at residue 366. In oneembodiment, the antibody or antibody fragment binding affinity for thevariant PCSK9 protein is at least 90% less relative to the wildtypeprotein of SEQ ID NO:755, wherein the variant protein comprises amutation at one or more of residue 147, 366 and 380. In a more specificembodiment, the mutation is one of S147F, E366K and V380M. In oneembodiment, the antibody or antibody fragment binding affinity for thevariant PCSK9 protein is at least 80% less relative to the wildtypeprotein of SEQ ID NO:755, wherein the variant protein comprises amutation at one or more of residue 147, 366 and 380. In a more specificembodiment, the mutation is one of S147F, E366K and V380M.

The invention encompasses anti-PCSK9 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful, or e.g., removal of afucose moiety to increase antibody dependent cellular cytotoxicity(ADCC) function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In a seventh aspect, the invention features a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds hPCSK9 and a pharmaceutically acceptable carrier. Inone embodiment, the invention features a composition which is acombination of an antibody or antigen-binding fragment of an antibody ofthe invention, and a second therapeutic agent. The second therapeuticagent may be any agent that is advantageously combined with the antibodyor fragment thereof of the invention, for example, an agent capable ofinducing a cellular depletion of cholesterol synthesis by inhibiting3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase, such as,for example, cerovastatin, atorvastatin, simvastatin, pitavastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.; capable ofinhibiting cholesterol uptake and or bile acid re-absorption; capable ofincreasing lipoprotein catabolism (such as niacin); and/or activators ofthe LXR transcription factor that plays a role in cholesterolelimination such as 22-hydroxycholesterol.

In an eighth aspect, the invention features methods for inhibitinghPCSK9 activity using the anti-PCSK9 antibody or antigen-binding portionof the antibody of the invention, wherein the therapeutic methodscomprise administering a therapeutically effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment of an antibody of the invention. The disorder treated is anydisease or condition which is improved, ameliorated, inhibited orprevented by removal, inhibition or reduction of PCSK9 activity.Specific populations treatable by the therapeutic methods of theinvention include subjects indicated for LDL apheresis, subjects withPCSK9-activating mutations (gain of function mutations, “GOF”), subjectswith heterozygous Familial Hypercholesterolemia (heFH); subjects withprimary hypercholesterolemia who are statin intolerant or statinuncontrolled; and subjects at risk for developing hypercholesterolemiawho may be preventably treated. Other indications include dyslipidemiaassociated with secondary causes such as Type 2 diabetes mellitus,cholestatic liver diseases (primary biliary cirrhosis), nephroticsyndrome, hypothyroidism, obesity; and the prevention and treatment ofatherosclerosis and cardiovascular diseases.

In specific embodiments of the method of the invention, the anti-hPCSK9antibody or antibody fragment of the invention is useful to reduceelevated total cholesterol, non-HDL cholesterol, LDL cholesterol, and/orapolipoprotein B (apolipoprotein B100).

The antibody or antigen-binding fragment of the invention may be usedalone or in combination with a second agent, for example, an HMG-CoAreductase inhibitor and/or other lipid lowering drugs.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Sequence comparison tables of heavy chain (A) and light chain(B) variable regions and CDRs of antibodies H1H316P and H1M300N.

FIG. 2. Antibody concentrations in serum over time. 316P 5 mg/kg (□);300N 5 mg/kg (◯); 316P 15 mg/kg (ν); 300N 15 mg/kg ().

FIG. 3. Serum total cholesterol level as a percentage of change overbuffer control. Buffer control (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ);316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

FIG. 4. Serum LDL cholesterol level as a percentage of change overbuffer control. Buffer Control (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ);316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

FIG. 5. Serum LDL cholesterol level normalized to buffer control. Buffercontrol (T); 316P 5 mg/kg (ν); 300N 5 mg/kg (σ): 316P 15 mg/kg (□); 300N15 mg/kg (Δ).

FIG. 6. Serum HDL cholesterol level as a percentage of change overbuffer control. Buffer control (T); 316P 5 mg/kg (ν): 300N 5 mg/kg (σ);316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

FIG. 7. Serum triglyceride level expressed as a percentage of changeover buffer control. Buffer control (T); 316P 5 mg/kg (ν); 300N 5 mg/kg(σ); 316P 15 mg/kg (□); 300N 15 mg/kg (Δ).

FIG. 8. Serum LDL cholesterol level expressed as a percentage of changeover baseline following a single dose subcutaneous administration. 316P5 mg/kg (ν); 300N 5 mg/kg ().

FIG. 9. Antibody concentrations in serum over time following a singledose subcutaneous administration, 316P 5 mg/kg (); 300N 5 mg/kg (σ).

FIG. 10. Western blot for mouse LDL receptor of total liver homogenates.Samples were taken 24 hours after PBS (lanes 1-3), 5 mg/kg 316P (lanes4-6), or 5 mg/kg of non-hPCSK9 specific mAb (lanes 7-8) administrationand 4 hours after 12 mg/kg hPCSK9-mmh (all lanes).

FIG. 11. Effects of 316P on serum LDL cholesterol level in PCSK9^(hu/hu)mice. Buffer control

316P1 mg/kg (

); 316P 5 mg/kg (

) 316P 10 mg/kg (

).

FIG. 12. Anti-hPCSK9 mAb serum pharmacokinetic profile in C57BL/6 mice.Single dose of Control I mAb (λ) at 10 mg/kg; 316P (σ) at 10 mg/kg and300N (ν) at 10 mg/kg.

FIG. 13. Anti-hPCSK9 mAb serum pharmacokinetic profile in hPCSK9heterozygous mice. Single dose of Control I mAb (λ) at 10 mg/kg; 316P(σ) at 10 mg/kg and 300N (ν) at 10 mg/kg.

FIG. 14. Effect of 316P on serum LDL cholesterol levels in SyrianHamster fed a normal diet. Buffer control (); 316P 1 mg/kg (ν); 316P 3mg/kg (σ); 316P 5 mg/kg (τ).

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

DEFINITIONS

The term “human proprotein convertase subtilisin/kexin type 9” or“hPCSK9”, as used herein, refers to hPCSK9 having the nucleic acidsequence shown in SEQ ID NO:754 and the amino acid sequence of SEC) IDNO:755, or a biologically active fragment thereof.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region (“HCVR”or “VH”) and a heavy chain constant region (comprised of domains CH1,CH2 and CH3). Each light chain is comprised of a light chain variableregion (“LCVR or “VL”) and a light chain constant region (CL). The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human mAbs of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antibody”, as usedherein, is not intended to include mAbs in which CDR sequences derivedfrom the germline of another mammalian species (e.g., mouse), have beengrafted onto human FR sequences.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by an equilibrium dissociation constant of at least about1×10⁻⁶ M or less (e.g., a smaller K_(D) denotes a tighter binding).Methods for determining whether two molecules specifically bind are wellknown in the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. An isolated antibody that specificallybinds hPCSK9 may, however, exhibit cross-reactivity to other antigenssuch as PCSK9 molecules from other species. Moreover, multi-specificantibodies (e.g., bispecifics) that bind to hPCSK9 and one or moreadditional antigens are nonetheless considered antibodies that“specifically bind” hPCSK9, as used herein.

The term “high affinity” antibody refers to those mAbs having a bindingaffinity to hPCSK9 of at least 10⁻¹⁰ M; preferably 10⁻¹¹ M; even morepreferably 10⁻¹² M, as measured by surface plasmon resonance, e.g.,BIACORE™ or solution-affinity ELISA.

By the term “slow off rate”, “Koff” or “kd” is meant an antibody thatdissociates from hPCSK9 with a rate constant of 1×10⁻³ s⁻¹ or less,preferably 1×10⁻⁴ s⁻¹ or less, as determined by surface plasmonresonance, e.g., BIACORE™.

The term “antigen-binding portion” of an antibody (or simply “antibodyfragment”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to hPCSK9. Anantibody fragment may include a Fab fragment, a F(ab′)₂ fragment, a Fvfragment, a dAb fragment, a fragment containing a CDR, or an isolatedCDR.

The specific embodiments, antibody or antibody fragments of theinvention may be conjugated to a therapeutic moiety (“immunoconjugate”),such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other mAbs having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hPCSK9 is substantially free of mAbs that specifically bindantigens other than hPCSK9). An isolated antibody that specificallybinds hPCSK9 may, however, have cross-reactivity to other antigens, suchas PCSK9 molecules from other species

A “neutralizing antibody”, as used herein (or an “antibody thatneutralizes PCSK9 activity”), is intended to refer to an antibody whosebinding to hPCSK9 results in inhibition of at least one biologicalactivity of PCSK9. This inhibition of the biological activity of PCSK9can be assessed by measuring one or more indicators of PCSK9 biologicalactivity by one or more of several standard in vitro or in vivo assaysknown in the art (see examples below).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” is a region of an antigen that is bound, by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.A “conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent or degree of similarity may beadjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Md. Biol. 24:307-331, which is herein incorporated by reference. Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartate andglutamate, and 7) sulfur-containing side chains: cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. (1992) Science 256: 1443 45, herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and (1997)Nucleic Acids Res. 25:3389 402, each of which is herein incorporated byreference.

In specific embodiments, the antibody or antibody fragment for use inthe method of the invention may be monospecific, bispecific, ormultispecific. Multispecific antibodies may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for epitopes of more than one target polypeptide. Anexemplary bi-specific antibody format that can be used in the context ofthe present invention involves the use of a first immunoglobulin (Ig)CH3 domain and a second Ig CH3 domain, wherein the first and second IgCH3 domains differ from one another by at least one amino acid, andwherein at least one amino acid difference reduces binding of thebispecific antibody to Protein A as compared to a bi-specific antibodylacking the amino acid difference. In one embodiment, the first Ig CH3domain binds Protein A and the second Ig CH3 domain contains a mutationthat reduces or abolishes Protein A binding such as an H95R modification(by IMGT axon numbering; H435R by EU numbering). The second CH3 mayfurther comprise an Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 mAbs; N44S, K52N, and V82I(IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 mAbs; andQ15R, N44S, KS2N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S,K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 mAbs.Variations on the bi-specific antibody format described above arecontemplated within the scope of the present invention.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known(see for example, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals,VELOCIMMUNE™). The VELOCIMMUNE™ technology involves generation of atransgenic mouse having a genome comprising human heavy and light chainvariable regions operably linked to endogenous mouse constant regionloci such that the mouse produces an antibody comprising a humanvariable region and a mouse constant region in response to antigenicstimulation. The DNA encoding the variable regions of the heavy andlight chains of the antibody are isolated and operably linked to DNAencoding the human heavy and light chain constant regions. The DNA isthen expressed in a cell capable of expressing the fully human antibody.In specific embodiment, the cell is a CHO cell.

Antibodies may be therapeutically useful in blocking a ligand-receptorinteraction or inhibiting receptor component interaction, rather than bykilling cells through fixation of complement and participation incomplement-dependent cytotoxicity (CDC), or killing cells throughantibody-dependent cell-mediated cytotoxicity (ADCC). The constantregion of an antibody is thus important in the ability of an antibody tofix complement and mediate cell-dependent cytotoxicity. Thus, theisotype of an antibody may be selected on the basis of whether it isdesirable for the antibody to mediate cytotoxicity.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an antibody molecule comprises a stablefour-chain construct of approximately 150-160 kDa in which the dimersare held together by an interchain heavy chain disulfide bond. In asecond form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, CH2 or CH3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

Generally, a VELOCIMMUNE™ mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. As described below, theantibodies are characterized and selected for desirable characteristics,including affinity, selectivity, epitope, etc. The mouse constantregions are replaced with a desired human constant region to generatethe fully human antibody of the invention, for example wild-type ormodified IgG1 or IgG4 (for example, SEQ ID NO:751, 752, 753). While theconstant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

Epitope Mapping and Related Technologies

To screen for antibodies that bind to a particular epitope (e.g., thosewhich block binding of IgE to its high affinity receptor), a routinecross-blocking assay such as that described Antibodies, Harlow and Lane(Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed.Other methods include alanine scanning mutants, peptide blots (Reineke(2004) Methods Mol Bid 248:443-63) (herein specifically incorporated byreference in its entirety), or peptide cleavage analysis. In addition,methods such as epitope excision, epitope extraction and chemicalmodification of antigens can be employed (Tomer (2000) Protein Science9: 487-496) (herein specifically incorporated by reference in itsentirety).

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (US2004/0101920, herein specifically incorporated by reference in itsentirety). Each category may reflect a unique epitope either distinctlydifferent from or partially overlapping with epitope represented byanother category. This technology allows rapid filtering of geneticallyidentical mAbs, such that characterization can be focused on geneticallydistinct mAbs. When applied to hybridoma screening, MAP may facilitateidentification of rare hybridoma clones that produce mAbs having thedesired characteristics. MAP may be used to sort the anti-PCSK9 mAbs ofthe invention into groups of mAbs binding different epitopes.

In various embodiments, the anti-hPCSK9 antibody or antigen-bindingfragment of an antibody binds an epitope within the catalytic domain,which is about 153 to 425 of SEQ ID NO:755); more specifically, anepitope from about 153 to about 250 or from about 250 to about 425; morespecifically, the antibody or antibody fragment of the invention bindsan epitope within the fragment from about 153 to about 208, from about200 to about 260, from about 250 to about 300, from about 275 to about325, from about 300 to about 360, from about 350 to about 400, and/orfrom about 375 to about 425.

In various embodiments, the anti-hPCSK9 antibody or antigen-bindingfragment of an antibody binds an epitope within the propeptide domain(residues 31 to 152 of SEQ ID NO:755); more specifically, an epitopefrom about residue 31 to about residue 90 or from about residue 90 toabout residue 152; more specifically, the antibody or antibody fragmentof the invention binds an epitope within the fragment from about residue31 to about residue 60, from about residue 60 to about residue 90, fromabout residue 85 to about residue 110, from about residue 100 to aboutresidue 130, from about residue 125 to about residue 150, from aboutresidue 135 to about residue 152, and/or from about residue 140 to aboutresidue 152.

In some embodiments, the anti-hPCSK9 antibody or antigen-bindingfragment of an antibody binds an epitope within the C-terminal domain,(residues 426 to 692 of SEQ ID NO:755); more specifically, an epitopefrom about residue 426 to about residue 570 or from about residue 570 toabout residue 692; more specifically, the antibody or antibody fragmentof the invention binds an epitope within the fragment from about residue450 to about residue 500, from about residue 500 to about residue 550,from about residue 550 to about residue 600, and/or from about residue600 to about residue 692.

In some embodiments, the antibody or antibody fragment binds an epitopewhich includes more than one of the enumerated epitopes within thecatalytic, propeptide or C-terminal domain, and/or within two or threedifferent domains (for example, epitopes within the catalytic andC-terminal domains, or within the propeptide and catalytic domains, orwithin the propeptide, catalytic and C-terminal domains.

In some embodiments, the antibody or antigen-binding fragment binds anepitope on hPCSK9 comprising amino acid residue 238 of hPCSK9 (SEQ IDNO:755). Experimental results (Table 27) show that when D238 wasmutated, the K_(D) of mAb 316P exhibited >400-fold reduction in bindingaffinity (˜1×10⁻⁹ M to ˜410×10⁻⁹ M) and T_(1/2) decreased >30-fold (from˜37 to ˜1 min). In a specific embodiment, the mutation was D238R. Inspecific embodiments, the antibody or antigen-binding fragment of theinvention binds an epitope of hPCSK9 comprising two or more of aminoacid residues at positions 153, 159, 238 and 343.

As shown below, a mutation in amino acid residue 153, 159 or 343resulted in about a 5- to 10-fold decrease in affinity or similarshortening in T_(1/2). In specific embodiments, the mutation was S153R,E159R and/or 0343R.

In some embodiments, the antibody or antigen-binding fragment binds anepitope on hPCSK9 comprising amino acid residue 366 of hPCSK9 (SEQ IDNO:755). Experimental results (Table 27) show that when E366 wasmutated, the affinity of mAb 300N exhibited about 50-fold decrease(˜0.7×10⁻⁹ M to ˜36×10⁻⁹ M) and a similar shortening in T_(1/2) (from˜120 to ˜2 min). In a specific embodiment, the mutation is E366K.

The present invention includes anti-PCSK9 antibodies that bind to thesame epitope as any of the specific exemplary antibodies describedherein. Likewise, the present invention also includes anti-PCSK9antibodies that compete for binding to PCSK9 or a PCSK9 fragment withany of the specific exemplary antibodies described herein.

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-PCSK9 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-PCSK9antibody of the invention, the reference antibody is allowed to bind toa PCSK9 protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the PCSK9 molecule is assessed. Ifthe test antibody is able to bind to PCSK9 following saturation bindingwith the reference anti-PCSK9 antibody, it can be concluded that thetest antibody binds to a different epitope than the reference anti-PCSK9antibody. On the other hand, if the test antibody is not able to bind tothe PCSK9 molecule following saturation binding with the referenceanti-PCSK9 antibody, then the test antibody may bind to the same epitopeas the epitope bound by the reference anti-PCSK9 antibody of theinvention.

To determine if an antibody competes for binding with a referenceanti-PCSK9 antibody, the above-described binding methodology isperformed in two orientations. In a first orientation, the referenceantibody is allowed to bind to a PCSK9 molecule under saturatingconditions followed by assessment of binding of the test antibody to thePCSK9 molecule. In a second orientation, the test antibody is allowed tobind to a PCSK9 molecule under saturating conditions followed byassessment of binding of the reference antibody to the PCSK9 molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the PCSK9 molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to PCSK9.As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50% but preferably 75%, 90% or even 99%as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have thesame epitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody reduce or eliminate bindingof the other. Two antibodies have overlapping epitopes if some aminoacid mutations that reduce or eliminate binding of one antibody reduceor eliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

In a specific embodiment, the invention comprises an anti-PCSK9 antibodyor antigen binding fragment of an antibody that binds an PCSK9 proteinof SEQ ID NO:755, wherein the binding between the antibody or fragmentthereof to PCSK9 and a variant PCSK9 protein is less than 50% of thebinding between the antibody or fragment and the PCSK9 protein of SEQ IDNO:755. In one specific embodiment, the variant PCSK9 protein comprisesat least one mutation of a residue at a position selected from the groupconsisting of 153, 159, 238 and 343. In a more specific embodiment, theat least one mutation is S153R, E159R, D238R, and/or D343R. In anotherspecific embodiment, the variant PCSK9 protein comprises at least onemutation of a residue at a position selected from the group consistingof 366. In one specific embodiment, the variant PCSK9 protein comprisesat least one mutation of a residue at a position selected from the groupconsisting of 147, 366 and 380. In a more specific embodiment, themutation is S147F, E366K and V380M.

Immunoconjugates

The invention encompasses a human anti-PCSK9 monoclonal antibodyconjugated to a therapeutic moiety (“immunoconjugate”), such as acytotoxin, a chemotherapeutic drug, an immunosuppressant or aradioisotope. Cytotoxin agents include any agent that is detrimental tocells. Examples of suitable cytotoxin agents and chemotherapeutic agentsfor forming immunoconjugates are known in the art, see for example. WO05/103081.

Bispecifics

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific mAbs may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al. (1991) J. Immunol. 147:60-69. The human anti-PCSK9 mAbs can belinked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment, to produce abispecific or a multispecific antibody with a second bindingspecificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)CH3 domain and a second Ig CH3 domain, wherein the first and second IgCH3 domains differ from one another by at least one amino acid, andwherein at least one amino acid difference reduces binding of thebispecific antibody to Protein A as compared to a bi-specific antibodylacking the amino acid difference. In one embodiment, the first Ig CH3domain binds Protein A and the second Ig CH3 domain contains a mutationthat reduces or abolishes Protein A binding such as an H95R modification(by IMGT exon numbering; H435R by EU numbering). The second CH3 mayfurther comprise a Y96F modification (by IMGT; Y436F by EU). Furthermodifications that may be found within the second CH3 include: D16E,L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N,V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, andV82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT;Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the caseof IgG4 antibodies. Variations on the bi-specific antibody formatdescribed above are contemplated within the scope of the presentinvention.

Bioequivalents

The anti-PCSK9 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described mAbs, but that retain the ability to bind humanPCSK9. Such variant mAbs and antibody fragments comprise one or moreadditions, deletions, or substitutions of amino acids when compared toparent sequence, but exhibit biological activity that is essentiallyequivalent to that of the described mAbs. Likewise, the anti-PCSK9antibody-encoding DNA sequences of the present invention encompasssequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-PCSK9 antibody or antibody fragment that isessentially bioequivalent to an anti-PCSK9 antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied. In one embodiment, two antigen-binding proteins arebioequivalent if there are no clinically meaningful differences in theirsafety, purity, and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in viva test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-PCSK9 antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation.

Treatment Population

The methods of the present invention comprise administering to a subjectin need thereof a therapeutic composition comprising an anti-PCSK9antibody. The therapeutic composition can comprise any of the anti-PCSK9antibodies, or fragments thereof, as disclosed herein. As used herein,the expression “a subject in need thereof” means a human or non-humananimal that exhibits one or more symptoms or indicia ofhypercholesterolemia or who has been diagnosed withhypercholesterolemia. Specific exemplary populations treatable by thetherapeutic methods of the invention include patients indicated for LDLapheresis, subjects with PCSK9-activating (GOF) mutations, patients withheterozygous Familial Hypercholesterolemia (heFH); subjects with primaryhypercholesterolemia who are statin intolerant or statin uncontrolled;and subjects at risk for developing hypercholesterolemia who may bepreventably treated.

While modifications in lifestyle and conventional drug treatment areoften successful in reducing cholesterol levels, not all patients areable to achieve the recommended target cholesterol levels with suchapproaches. Various conditions, such as familial hypercholesterolemia(FH), appear to be resistant to lowering of LDL-C levels in spite ofaggressive use of conventional therapy. Homozygous and heterozygousfamilial hypercholesterolemia (hoFH, heFH) are conditions associatedwith premature atherosclerotic vascular disease. However, patientsdiagnosed with hoFH are largely unresponsive to conventional drugtherapy and have limited treatment options. Specifically, treatment withstatins, which reduce LDL-C by inhibiting cholesterol synthesis andupregulating the hepatic LDL receptor, may have little effect inpatients whose LDL receptors are non-existent or defective. A mean LDL-Creduction of only less than about 20% has been recently reported inpatients with genotype-confirmed hoFH treated with the maximal dose ofstatins. The addition of ezetimibe 10 mg/day to this regimen resulted ina total reduction of LDL-C levels of 27%, which is still far fromoptimal. Likewise, many patients are statin non-responsive, poorlycontrolled with statin therapy, or cannot tolerate statin therapy; ingeneral, these patients are unable to achieve cholesterol control withalternative treatments. There is a large unmet medical need for newtreatments that can address the short-comings of current treatmentoptions.

Thus, the invention includes therapeutic methods in which the antibodyor antibody fragment of the invention is administered to a patient totreat hypercholesterolemia. Specific non-limiting examples of types ofhypercholesterolemia which are treatable in accordance with the methodsof the present invention include, e.g., heterozygous FamilialHypercholesterolemia (heFH), homozygous Familial Hypercholesterolemia(hoFH), as well as incidences of hypercholesterolemia that are distinctfrom Familial Hypercholesterolemia (nonFH).

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising theanti-PCSK9 antibodies or antigen-binding fragments thereof of thepresent invention. The administration of therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™) DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose may vary depending upon the age and the size of a subject to beadministered, target disease, conditions, route of administration, andthe like. When the antibody of the present invention is used fortreating various conditions and diseases associated with PCSK9,including hypercholesterolemia, disorders associated with LDL andapolipoprotein B, and lipid metabolism disorders, and the like, in anadult patient, it is advantageous to intravenously administer theantibody of the present invention normally at a single dose of about0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.Depending on the severity of the condition, the frequency and theduration of the treatment can be adjusted.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticies, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen. HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

Dosage

The amount of anti-PCSK9 antibody administered to a subject according tothe methods of the present invention is, generally, a therapeuticallyeffective amount. As used herein, the phrase “therapeutically effectiveamount” means a dose of anti-PCSK9 antibody that results in a detectableimprovement in one or more symptoms or indicia of hypercholesterolemia,or a dose of anti-PCSK9 antibody that inhibits, prevents, lessens, ordelays the progression of hypercholesterolemia in a patient. In the caseof an anti-PCSK9 antibody, a therapeutically effective amount can befrom about 0.05 mg to about 600 mg, e.g., about 0.05 mg, about 0.1 mg,about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg,about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570mg, about 580 mg, about 590 mg, or about 600 mg, of the anti-PCSK9antibody.

The amount of anti-PCSK9 antibody contained within the individual dosesmay be expressed in terms of milligrams of antibody per kilogram ofpatient body weight (i.e., mg/kg). For example, the anti-PCSK9 antibodymay be administered to a patient at a dose of about 0.0001 to about 10mg/kg of patient body weight.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of anti-PCSK9 antibody may be administered to a subject over adefined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of anti-PCSK9 antibody. As used herein, “sequentiallyadministering” means that each dose of anti-PCSK9 antibody isadministered to the subject at a different point in time, e.g., ondifferent days separated by a predetermined interval (e.g., hours, days,weeks or months). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of ananti-PCSK9 antibody, followed by one or more secondary doses of theanti-PCSK9 antibody, and optionally followed by one or more tertiarydoses of the anti-PCSK9 antibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-PCSK9 antibody.Thus, the “initial dose” is the dose which is administered at thebeginning of the treatment regimen (also referred to as the “baselinedose”); the “secondary doses” are the doses which are administered afterthe initial dose; and the “tertiary doses” are the doses which areadministered after the secondary doses. The initial, secondary, andtertiary doses may all contain the same amount of anti-PCSK9 antibody,but will generally differ from one another in terms of frequency ofadministration. In certain embodiments, however, the amount ofanti-PCSK9 antibody contained in the initial, secondary and/or tertiarydoses will vary from one another (e.g., adjusted up or down asappropriate) during the course of treatment.

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 30 (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, or more) days after the immediatelypreceding dose, or 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,or more) weeks after the immediately preceding dose. The phrase “theimmediately preceding dose,” as used herein, means, in a sequence ofmultiple administrations, the dose of anti-PCSK9 antibody which isadministered to a patient prior to the administration of the very nextdose in the sequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-PCSK9 antibody. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to12 weeks after the immediately preceding dose (e.g., once every week[Q1W], once every two weeks [Q2W], once every three weeks [Q3W], onceevery four weeks [Q4W], once every six weeks [Q6W], once every eightweeks [Q8W], etc.). Similarly, in embodiments involving multipletertiary doses, each tertiary dose may be administered at the samefrequency as the other tertiary doses. For example, each tertiary dosemay be administered to the patient 1 to 12 weeks after the immediatelypreceding dose (e.g., once every week [Q1W], once every two weeks [Q2W],once every three weeks [Q3W], once every four weeks [Q4W], once everysix weeks [Q6W], once every eight weeks [Q8W], etc.). Alternatively, thefrequency at which the secondary and/or tertiary doses are administeredto a patient can vary over the course of the treatment regimen. Thefrequency of administration may also be adjusted during the course oftreatment by a physician depending on the needs of the individualpatient following clinical examination.

Non-limiting exemplary administration regimens of the present inventioninclude, e.g., 75 mg of anti-PCSK9 antibody (e.g., 300N or 316P)administered to a subject once every two weeks (Q2W); 100 mg ofanti-PCSK9 antibody (e.g., 300N or 316P) administered to a subject onceevery two weeks (Q2W), and 150 mg of anti-PCSK9 antibody (e.g., 300N or316P) administered to a subject once every two weeks (Q2W).

The present invention also includes administration regimens comprisingadministering one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or more) doses comprising 75 mg of anti-PCSK9 antibody (e.g.,300N or 316P) to a patient, and if the patient has not achieved asatisfactory reduction in LDL-C following administration of one or moreof the 75 mg doses (or if an increased dose is otherwise deemed moretherapeutically appropriate), then discontinuing the 75 mg doses andadministering one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or more) additional doses comprising 150 mg of the anti-PCSK9antibody to the patient, wherein each 75 mg and 150 mg dose isadministered to the patient once every two weeks (i.e., Q2W dosing). Asused herein, the phrase “satisfactory reduction in LDL-C” means that theblood concentration of LDL-C in the patient following administration ofone or more doses of anti-PCSK9 antibody is less than about 100 mg/dL orless than about 70 mg/dL.

The present invention also includes administration regimens comprisingadministering one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or more) doses comprising 150 mg of anti-PCSK9 antibody (e.g.,300N or 316P) to a patient, followed by discontinuing the 150 mg dosesand instead administering one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or more) additional doses comprising 75 mg of theanti-PCSK9 antibody to the patient, wherein each 150 mg and 75 mg doseis administered to the patient once every two weeks (i.e. Q2W dosing).

The present invention also includes administration regimens comprisingadministering one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or more) doses comprising 75 mg of anti-PCSK9 antibody (e.g.,300N or 316P) to a patient, followed by discontinuing the 75 mg dosesand instead administering one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or more) additional doses comprising 150 mg of theanti-PCSK9 antibody to the patient, followed by discontinuing the 150 mgdoses and resuming administration of one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more) additional doses comprising 75mg of the anti-PCSK9 antibody to the patient, wherein each 150 mg and 75mg dose is administered to the patient once every two weeks (i.e., Q2Wdosing).

Combination and Adjunct Therapies

The methods of the present invention, according to certain embodiments,may comprise administering a pharmaceutical composition comprising ananti-PCSK9 antibody to a patient who is on a therapeutic regimen for thetreatment of hypercholesterolemia at the time of, or just prior to,administration of the pharmaceutical composition of the invention. Forexample, a patient who has previously been diagnosed withhypercholesterolemia may have been prescribed and is taking a stabletherapeutic regimen of another drug prior to and/or concurrent withadministration of a pharmaceutical composition comprising an anti-PCSK9antibody. The prior or concurrent therapeutic regimen may comprise,e.g., (1) an agent which induces a cellular depletion of cholesterolsynthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A(CoA) reductase, such as a statin (e.g., cerivastatin, atorvastatin,simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin,pravastatin, etc.); (2) an agent which inhibits cholesterol uptake andor bile acid re-absorption; (3) an agent which increase lipoproteincatabolism (such as niacin); and/or (4) activators of the LXRtranscription factor that plays a role in cholesterol elimination suchas 22-hydroxycholesterol. In certain embodiments, the patient, prior toor concurrent with administration of an anti-PCSK9 antibody is on afixed combination of therapeutic agents such as ezetimibe plussimvastatin; a statin with a bile resin (e.g., cholestyramine,colestipol, colesevelam); niacin plus a statin (e.g., niacin withlovastatin); or with other lipid lowering agents such as omega-3-fattyacid ethyl esters for example, omacor).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human PCSK9

VELOCIMMUNE™ mice were immunized with human PCSK9, and the antibodyimmune response monitored by antigen-specific immunoassay using serumobtained from these mice. Anti-hPCSK9 expressing B cells were harvestedfrom the spleens of immunized mice shown to have elevated anti-hPCSK9antibody titers were fused with mouse myeloma cells to form hybridomas.The hybridomas were screened and selected to identify cell linesexpressing hPCSK9-specific antibodies using assays as described below.The assays identified several cell lines that produced chimericanti-hPCSK9 antibodies designated as H1M300, H1M504, H1M505, H1M500,H1M497, H1M498, H1M494, H1M309, H1M312, H1M499, H1M493, H1M496, H1M503,H1M502, H1M508, H1M495 and H1M492.

Human PCSK9-specific antibodies were also isolated directly fromantigen-immunized B cells without fusion to myeloma cells, as describedin U.S. 2007/0280945A1, hereby incorporated by reference in itsentirety. Heavy and light chain variable regions were cloned to generatefully human anti-hPCSK9 antibodies designated as H1H313, H1H314, H1H315,H1H316, H1H317, H1H318, H1H320, H1H321 and H1H334. Stable recombinantantibody-expressing CHO cell lines expressing these antibodies wereestablished.

Example 2 Gene Utilization Analysis

To analyze the structure of the mAbs produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. Thepredicted amino acid sequences of the variable regions were confirmed byN-terminal amino acid sequencing. From the nucleic acid sequence andpredicted amino acid sequence of the mAbs, gene usage was identified foreach antibody chain.

TABLE 1 Heavy Chain Light Chain Variable Region Variable Region AntibodyVH D JH VK JK H1H313 3-13 1-26 4 3-15 3 H1H314 3-33 3-3  4 1-5  2 H1H3153-33 3-3  4 4-1  1 H1H316 3-23 7-27 2 4-1  2 H1H317 3-13 1-26 4 1-6  1H1H318 4-59 3-10 6 1-9  1 H1H320 1-18 2-2  6 2-30 1 H1H321 2-5  1-7  62-28 4 H1H334 2-5  6-6  6 2-28 4 H1M300 3-7  2-8  6 2-28 4 H1M504 3-302-8  6 2-28 4 H1M505 3-30 2-8  6 2-28 4 H1M500 2-5  5-5  6 2-28 4 H1M4971-18 2-2  6 2-30 2 H1M498 3-21 2-2  4 1-5  2 H1M494 3-11 5-12 6 3-20 4H1M309 3-21 6-13 4 1-5  1 H1M312 3-21 6-13 4 1-5  1 H1M499 3-21 6-13 41-5  1 H1M493 3-21 6-13 4 1-5  1 H1M496 3-13 6-19 4 3-15 3 H1M503 1-182-2  6 2-28 1 H1M502 3-13 6-13 4 3-15 3 H1M508 3-13 6-13 4 3-15 3 H1M4953-9  4-17 6 1-9  3 H1M492 3-23 3-3  2 3-20 4

Example 3 Antigen Binding Affinity Determination

Equilibrium dissociation constants (K_(D)) for hPCSK9 binding to mAbsgenerated by hybridoma cell lines described above were determined bysurface kinetics in a real-time biosensor surface plasmon resonanceassay (BIACORE™ T100). Each antibody was captured at a flow rate of 4μl/min for 90 sec on a goat anti-mouse IgG polyclonal antibody surfacecreated through direct chemical coupling to a BIACORE™ chip to form acaptured antibody surface. Human PCSK9-myc-myc-his (hPCSK9-mmh) at aconcentration of 50 nM or 12.5 nM was injected over the capturedantibody surfaces at a flowrate of 50 μl/min for 300 sec, andantigen-antibody dissociation was monitored for 15 min at either 25° C.or 37° C. (K_(D)=pM; T_(1/2)=min).

TABLE 2 25° C. 37° C. Antibody K_(D) T_(1/2) K_(D) T_(1/2) H1M300 399170 1510 32 H1M309 29.9 7461 537 326 H1M312 0.225 15568 432 392 H1M49346.5 4921 522 341 H1M494 870 114 2350 30 H1M495 440 222 7500 19 H1M496254 257 421 118 H1M497 20.1 5801 480 290 H1M498 6400 30 7500 14 H1M499106 2253 582 316 H1M500 1400 91 6010 15 H1M502 78.3 958 411 151 H1M503510 118 1880 30 H1M504 3470 35 11200 6 H1M505 2740 42 9200 6 H1M508 138572 442 139 H1M510 1070 68 3960 10

Equilibrium dissociation constants (K_(D)) for hPCSK9 binding to mAbsgenerated via direct isolation of splenocytes were determined by surfacekinetics in a real-time biosensor surface plasmon resonance assay(BIACORE™ T100). Each selected antibody was captured at a flowrate of 2μl/min for 6 min on a goat anti-human IgG polyclonal antibody surfacecreated through direct chemical coupling to a BIACORE™ chip to form acaptured antibody surface. Human PCSK9-mmh at a concentration of 50 nMor 12.5 nM was injected over the captured antibody surface at a flowrateof 70 μl/min for 5 min, and antigen-antibody dissociation was monitoredfor 15 min at either 25° C. or 37° C. (K_(D)=pM; T_(1/2)=min).

TABLE 3 25° C. 37° C. Antibody K_(D) T_(1/2) K_(D) T_(1/2) H1H313P 244230 780 60 H1H314P 3990 65 3560 43 H1H315P 129 151 413 35 H1H316P 377 421080 11 H1H317P 30400 137 18600 70 H1H318P 972 59 1690 28 H1H320P 771 281930 8 H1H321P 865 106 3360 23 H1H334P 3750 46 15900 8

Dissociation rate (kd) of selected mAbs for tagged rhesus monkey (Macacamulata) PCSK9 (mmPCSK9; SEQ ID NO:756) (mmPCSK9-mmh) at 25° C. wasdetermined as described above.

TABLE 4 Antibody kd (1/s) T_(1/2) (min) H1H313P 2.92 × 10⁻⁵ 396 H1H318P3.69 × 10⁻³ 3 H1H334P 8.06 × 10⁻³ 1 H1H315P 2.29 × 10⁻⁴ 51 H1H316P 2.29× 10⁻⁴ 51 H1H320P 3.17 × 10⁻⁴ 36 H1M300 1.52 × 10⁻⁴ 76 H1M504 5.04 ×10⁻⁴ 23 H1M497 6.60 × 10⁻⁵ 175 H1M503 8.73 × 10⁻⁵ 132 H1M496 4.45 × 10⁻⁵260

Example 4 Effect of pH on Antigen Binding Affinity

The effects of pH on antigen binding affinity for CHO cell-producedfully human a hPCSK9 mAbs was assessed as described above. The mAbstested are fully human versions of H1H316P (“316P”) (HCVR/LCVR SEQ IDNO: 90/92; CDR sequences SEQ ID NO: 76/78/80 and 84/86/88) and H1M300N(“300N”) (HCVR/LCVR SEQ ID NO: 218/226; CDR sequences SEQ IDNO:220/222/224 and 228/230/232). Human PCSK9-myc-myc-his (hPCSK9-mmh)was captured on an anti-myc mAb surface either at a high density (about35 to 45 resonance units) (RU) or at a low density (about 5 to 14 RU).Each antibody, at 50 nM in HBST (pH 7.4 or pH 5.5) was injected over thecaptured hPCSK9 surface at a flow rate of 100 μl/ml for 1.5 min at 25°C. and antigen-antibody dissociation was monitored for 10 min, ControlI: anti-hPCSK9 mAb SEQ ID NO:79/101 (WO 2008/063382) (K_(D)=pM;T_(1/2)=min).

TABLE 5 High hPCSK9 Low hPCSK9 Density Surface Density Surface pH 7.4 pH5.5 pH 7.4 pH 5.5 Antibody K_(D) T_(1/2) K_(D) T_(1/2) K_(D) T_(1/2)K_(D) T_(1/2) 316P 191 74  144 83 339  45  188 58 300N 65 507 1180 26310 119 1380 13 Control I 20000 29 ND ND ND ND ND ND

The antigen binding properties of 316P and 300N at pH 7.4 or pH 5.5 weredetermined by a modified BIACORE™ assay as described above. Briefly,mAbs were immobilized onto BIACORE™ CM5 sensor chips via amine coupling.Varying concentrations of myc-myc-his tagged hPCSK9, mouse PCSK9(mPCSK9, SEQ ID NO:757), hPCSK9 with a gain of function (GOF) pointmutation of D374Y (hPCSK9(D374Y), cynomolgus monkey (Macacafascicularis) PCSK9 (mfPCSK9, SEQ ID NO:761) (mfPCSK9), rat (Rattusnorvegicus) PCSK9 (rPCSK9, SEQ ID NO:763), and his-tagged Syrian goldenhamster (Mesocricetus auratus) PCSK9 (maPCSK9, SEQ ID NO:762) (maPCSK9),ranging from 11 to 100 nM, were injected over the antibody surface atthe flow rate of 100 μl/ml for 1.5 min and antigen-antibody dissociationwas monitored in real time for 5 min at either 25° C. (Table 6) or 37°C. (Table 7). Control II: anti-hPCSK9 mAbs SEQ ID NO:67/12 (WO2009/026558) (NB: no binding was observed under the experimentalcondition) (K_(D)=pM, T_(1/2)=min).

TABLE 6 pH Effect at 25° C. pH 7.4 pH 5.5 Antigen K_(D) T_(1/2) K_(D)T_(1/2) 316P hPCSK9-mmh 1260 36 22 39 mPCSK9-mmh 4460 10 63 11hPCSK9(D347Y)-mmh 2490 15 166 13 mfPCSK9-mmh 1420 42 8 23 maPCSK9-h 83508 87 8 rPCSK9-mmh 24100 2 349 5 300N hPCSK9-mmh 1100 76 3100 5mPCSK9-mmh NB NB NB NB hPCSK9(D347Y)-mmh 1310 46 9030 3 mfCSK9-mmh 217031 38500 0.4 maPCSK9-h NB NB NB NB rPCSK9-mmh NB NB NB NB Control IhPCSK9-mmh 33100 14 1740 31 mPCSK9-mmh NB NB NB NB hPCSK9(D347Y)-mmh71000 11 7320 30 mfPCSK9-mmh 362000 0.2 67200 3 maPCSK9-h NB NB NB NBrPCSK9-mmh NB NB NB NB Control II hPCSK9-mmh 143 266 2 212 mPCSK9-mmh3500 11 33 12 hPCSK9(D347Y)-mmh 191 155 49 56 mfPCSK9-mmh 102 262 12 63maPCSK9-h 6500 3 ND ND rPCSK9- mmh 22400 2 106 5

TABLE 7 pH Effect at 37° C. pH 7.4 pH 5.5 Antigen K_(D) T_(1/2) K_(D)T_(1/2) 316P hPCSK9-mmh 4000 9 142 11 mPCSK9-mmh 12200 3 13600 3hPCSK9(D347Y)-mmh 6660 4 1560 5 mfPCSK9-mmh 3770 11 44 5 maPCSK9-h 217002 ND ND rPCSK9-mmh 55100 2 399 1 300N hPCSK9-mmh 2470 20 11900 1mPCSK9-mmh NB NB NB NB hPCSK9(D347Y)-mmh 2610 14 28000 1 mfPCSK9-mmh4810 8 65200 0.1 maPCSK9-h NB NB NB NB rPCSK9-mmh NB NB NB NB Control IhPCSK9-mmh 45900 0.1 11300 3 mPCSK9-mmh NB NB NB NB hPCSK9(D347Y)-mmh169000 0.4 27000 3 mfPCSK9-mmh 500000 0.6 5360 0.3 maPCSK9-h NB NB NB NBrPCSK9 NB NB NB NB Control II hPCSK9-mmh 284 87 20 44 mPCSK9-mmh 8680 389 3 hPCSK9(D347Y)-mmh 251 57 483 26 mfPCSK9-mmh 180 127 214 65maPCSK9-h 8830 0.5 ND ND rPCSK9p-mmh 30200 1 233 1

Example 5 Anti-hPCSK9 mAbs Binding to hPCSK9 with Point Mutation D374Y

The binding affinity of selected anti-hPCSK9 mAbs to hPCSK9 with a gainof function (GOF) point mutation of D374Y (hPCSK9(D374Y)-mmh) wasdetermined as described above. Each antibody was captured at a flowrateof 40 μl/min for 8-30 sec on a goat anti-human IgG polyclonal antibodysurface created through direct chemical coupling to a BIACORE™ chip toform a captured antibody surface. hPCSK9(D374Y)-mmh at varyingconcentrations of 1.78 nM to 100 nM was injected over the capturedantibody surface at a flowrate of 50 μl/min for 5 min, and thedissociation of hPCSK9(D374Y)-mmh and antibody was monitored for 15 minat 25° C. Control III: anti-hPCSK9 mAbs SEQ ID NO:49/23 (WO 2009/026558)(K_(D)=pM; T_(1/2)=min).

TABLE 8 Antibody K_(D) T_(1/2) 316P 1780 14 300N 1060 49 Control I 2360025 Control II 66 216 Control III 1020 126

Example 6 Binding Specificity of Anti-hPCSK9 mAbs

316P, 300N, and Control I anti-hPCSK9 mAbs were captured on namine-coupled anti-hFc CM5 chip on BIACORE™2000. Tagged (myc-myc-his)human PCSK9, human PCSK1 (hPCSK1) (SEQ ID NO:759), human PCSK7 (hPCSK7)(SEQ ID NO:760), or mouse PCSK9 were injected (100 nM) over the capturedmAb surface and allowed to bind at 25° C. for 5 min. Changes in RU wererecorded. Results: 300N and Control I bound only to hPCSK9, and 316Pbound both hPCSK9 and mPCSK9.

The binding specificities of anti-hPCSK9 mAbs were determined by ELISA.Briefly, anti-hPCSK9 antibody was coated on a 96-well plate. HumanPCSK9-mmh, mPCSK9-mmh, maPCSK9-h, hPCSK1-mmh, or hPCSK7-mmh, at 1.2 nM,were added to antibody-coated plates and incubated at RT for 1 hr.Plate-bound PCSK protein was then detected by HRP-conjugated anti-Hisantibody. Results show that 316P binds human, mouse, and hamster PCSK9,whereas 300N and Control. I only bound hPCSK9. None of the anti-hPCSK9mAbs exhibited significant binding to hPCSK1 or hPCSK7.

Example 7 Cross-Reactivity of Anti-hPCSK9 mAbs

Cross-reactivity of anti-hPCSK9 mAbs with mmPCSK9, mfPCSK9, mPCSK9,maPCSK9, or rPCSK9 was determined using BIACORE™3000. Briefly,anti-hPCSK9 mAbs were captured on an anti-hFc surface created throughdirect chemical coupling to a BIACORE™ chip. Purified tagged hPCSK9,hPCSK9(D374Y), mmPCSK9, mfPCSK9, mPCSK9, maPCSK9, or rPCSK9, each at1.56 nM to 50 nM, was injected over the antibody surface at either 25°C. or 37° C. Binding between 316P, 300N, Control I, Control II, orControl III and the PCSK9 proteins was determined (K_(D)=pM;T_(1/2)=min) (ND=not determined).

TABLE 9 316P mAb 37° C. 25° C. Antigen K_(D) T_(1/2) K_(D) T_(1/2)hPCSK9-mmh 1800 9 580 36 hPCSK9(D374Y)-mmh 4200 4 1690 15 mmPCSK9-mmh1800 21 550 92 mfPCSK9-mmh 1800 11 520 60 mPCSK9-mmh 4700 3 2300 11maPCSK9-h 19000 1 6810 5 rPCSK9-mmh 37500 1 14500 2

TABLE 10 300N mAb 37° C. 25° C. Antigen K_(D) T_(1/2) K_(D) T_(1/2)hPCSK9-mmh 2400 22 740 110 hPCSK9(D374Y)-mmh 2200 14 900 65 mmPCSK9-mmh1600 26 610 79 mfPCSK9-mmh 3800 11 1500 45 mPCSK9-mmh NB NB NB NBmaPCSK9-h NB NB NB NB rPCSK9-mmh NB NB NB NB

TABLE 11 Control I mAb 37° C. 25° C. Antigen K_(D) T_(1/2) K_(D) T_(1/2)hPCSK9-mmh 226000 2 27500 16 hPCSK(D374Y)-mmh ND ND 23600 25 mmPCSK9-mmh420000 3 291000 2 mfPCSK9-mmh 14300 10 24900 14 mPCSK9-mmh NB NB NB NBmaPCSK9-h NB NB NB NB rPCSK9-mmh NB NB NB NB

TABLE 12 Control II mAb 37° C. 25° C. Antigen K_(D) T_(1/2) K_(D)T_(1/2) hPCSK9-mmh 91 162 61 372 hPCSK9(D374Y)-mmh 93 90 66 216mfPCSK9-mmh 33 252 26 546 mPCSK9-mmh 4700 3 2300 11 maPCSK9-h 60800 0.425000 2 rPCSK9-mmh 14100 1 6900 3

TABLE 13 Control III mAb 37° C. 25° C. Antigen K_(D) T_(1/2) K_(D)T_(1/2) hPCSK9-mmh 380 378 490 450 hPCSK(D374Y)-mmh 130 660 1000 126mfPCSK9-mmh 110 750 340 396 mPCSK9-mmh 33500 1 10900 4 maPCSK9-h 780 1072100 67 rPCSK9-mmh NB NB 33200 2

Example 8 Inhibition of Binding Between hPCSK9 and hLDLR Domains

The ability of selected anti-hPCSK9 mAbs to block hPCSK9 binding tohuman LDLR full length extracellular domain (hLDLR-ecto SEQ ID NO:758),hLDLR EGF-A domain (amino acids 313-355 of SED ID NO:758), or hLDLREGF-AB domains (amino acids of 314-393 of SEQ ID NO:758) (LDLR Genbanknumber NM_(—)000527) was evaluated using BIACORE™ 3000. Briefly,hLDLR-ecto, EGF-A-hFc, or EGF-AB-hFc protein was amine-coupled on a CM5chip to create a receptor or receptor fragment surface. Selectedanti-hPCSK9 mAbs, at 62.5 nM (2.5 fold excess over antigen), werepremixed with 25 nM of hPCSK9-mmh, followed by 40 min incubation at 25°C. to allow antibody-antigen binding to reach equilibrium to formequilibrated solutions. The equilibrated solutions were injected overthe receptor or receptor fragment surfaces at 2 μl/min for 40 min at 25°C. Changes in RU due to the binding of the anti-hPCSK9 mAbs tohLDLR-ecto, EGF-A-hFc, or EGF-AB-hFc were determined. Results show thatH1H316P and H1M300N blocked the binding of hPCSK9-mmh to hLDLR-ecto,hLDLR EGF-A domain, and hLDLR EGF-AB domains; H1H320P blocked thebinding of hPCSK9-mmh to hLDLR-ecto and hLDLR EGF-A domain; and H1H321Pblocked the binding of hPCSK9-mmh to hLDLR EGF-A domain.

The ability of the mAbs to block hPCSK9 binding to hLDLR-ecto, hLDLREGF-A domain, or hLDLR EGF-AB domains was also evaluated with anELISA-based immunoassay. Briefly, hLDLR-ecto, hLDLR EGF-A-hFc or hLDLREGF-AB-hFc, each at 2 μg/ml, was coated on a 96-well plate in PBS bufferovernight at 4° C., and nonspecific binding sites blocked with BSA. Thisplate was used to measure free hPCSK9-mmh in a PCSK9-mmh solutionpre-equilibrated with varying concentrations of anti-hPCSK9 mAbs. Aconstant amount of hPCSK9-mmh (500 pM) was pre-mixed with varied amountsof antibody, ranging from 0 to ˜50 nM serial dilutions, followed by 1 hrincubation at room temperature (RT) to allow antibody-antigen binding toreach equilibrium. The equilibrated sample solutions were transferred toreceptor or receptor fragment coated plates. After 1 hour of binding,the plates were washed and bound hPCSK9-mmh detected using HRPconjugated anti-myc antibody. IC₅₀ values (in pM) were determined as theamount of antibody required to achieve 50% reduction of hPCSK9-mmh boundto the plate-coated receptor or receptor fragment. The results show thatspecific mAbs functionally block PCSK9 from binding the three receptorsat both neutral pH (7.2) and acidic pH (5.5).

TABLE 14 pH 7.2 pH 5.5 Plate Coating Surface Ab hLDLR-ecto EGF-A EGF-ABhLDLR-ecto EGF-A EGF-AB 316P <125 <125 <125 <125 <125 <125 300N 144 146<125 1492 538 447 Control I — >100,000 >100,000 — >100,000 >100,000Control II 288 510 274 411 528 508 Control III 303 635 391 742 787 1073

The ability of the mAbs to block hPCSK9 GOF mutant hPCSK9(D374Y)-mmhbinding to hLDLR EGF-A domain or hLDLR EGF-AB domain (IC₅₀ values in pM)was also evaluated with the ELISA-based immunoassay described aboveusing a constant amount of 0.05 nM hPCSK9(D374Y)-mmh.

TABLE 15 pH 7.2 pH 5.5 Plate Coating Surface EGF-A EGF-AB EGF-A EGF-AB316P 203 139 1123 1139 300N 135 142 3463 3935 ControlI >100,000 >100,000 >100,000 >100,000 Control II 72 57 129 118 ControlIII 537 427 803 692

The ability of the mAbs to block either mmPCSK9 or mPCSK9 binding tohLDLR-ecto domain, hLDLR EGF-A domain, or hLDLR EGF-AB domain (IC₅₀values in pM) was evaluated at neutral pH (7.2) with the ELISA-basedimmunoassay describe above using a constant amount of 1 nM of mmh-taggedmmPCSK9 or 1 nM of mPCSK9.

TABLE 16 1 nM mmPCSK9-mmh 1 nM mPCSK9-mmh hLDLR-ecto EGF-A EGF-AB EGF-AEGF-AB 316P <250 <250 <250 <250 <250 300N 255 256 290 >33000 >33000

The ability of the mAbs to block hPCSK9, mmPCSK9, rPCSK9, maPCSK9,mfPCSK9, or mPCSK9 binding to hLDLR EGF-A domain (IC₅₀ values in pM) wasevaluated at neutral pH (7.2) (Table 17) acidic pH (5.5, Table 18) withthe ELISA-based immunoassay described above using a constant amount of0.5 nM of hPCSK9-mmh, 1 nM of mmPCSK9-mmh, 1 nM of rPCSK9-mmh, 1 nM ofmaPCSK9-h, 0.3 nM of mfPCSK9-mmh, or 1 nM of mPCSK9-mmh.

TABLE 17 hPCSK9 mmPCSK9 rPCSK9 maPCSK9 mfPCSK9 mPCSK9 316P <125 <2502662 349 75 305 300N 182 460 >100000 >100000 473 >100000 Control I— >100000 >100000 >100000 >100000 >100000 Control II 146 83 2572 2038361 855 Control III 249 293 >100000 245 572 >100000

TABLE 18 hPCSK9 mmPCSK9 rPCSK9 maPCSK9 mPCSK9 316P <125 <250 42880 1299991 300N 223 3704 >100000 >100000 >100000 ControlI >100000 >100000 >100000 >100000 >10000 Control II 154 <250 11640 83392826 Control III 390 376 >100000 414 >100000

The ability of 316P and Control I to block hPCSK9 binding to hLDLR wasalso determined. Briefly, either recombinant hLDLR or hLDLR-EGFA-mFc wasimmobilized onto BIACORE™ CM5 chips via amine coupling. Anantigen-antibody mixture of 100 nM hPCSK9-mmh and 316P, Control I mAb,or a non-hPCSK9 specific mAb (each at 250 nM) was incubated at RT for 1hr, and then injected over the hLDLR or hLDLR-EGFA surface at the flowrate of 10 μl/ml for 15 min at 25° C. Changes in RU due to the bindingbetween the free hPCSK9-mmh in the mixture to either hLDLR or hLDLR-EGFAwere recorded. The binding of hPCSK9 to either hLDLR or hLDLR-EGFA wascompletely blocked by 316P and 300N but not by Control I mAb.

Example 9 Epitope Mapping

In order to determine epitope-binding specificity, three chimericPCSK9-mmh proteins were generated in which specific human PCSK9 domainswere substituted with mouse PCSK9 domains. Chimeric protein #1 consistsof a mouse PCSK9 pro-domain (amino acid residues 1-155 of SEQ ID NO:757)followed by a human PCSK9 catalytic domain (residues 153-425 of SEQ IDNO:755) and a mouse PCSK9 C-terminal domain (residues 429-694 SEQ IDNO:757) (mPro-hCat-mC-term-mmh). Chimeric protein #2 consists of a humanPCSK9 pro-domain (residues 1-152 of SEQ ID NO:755) followed by a mousePCSK9 catalytic domain (residues 156-428 of SEQ ID NO:757) and a mousePCSK9 C-terminal (hPro-mCat-mC-term-mmh). Chimeric protein #3 consistsof mouse PCSK9 pro-domain and a mouse PCSK9 catalytic domain followed bya human PCSK9 C-terminal domain (residues 426-692 of SEQ ID NO:755)(mPro-mCat-hC-term-mmh). In addition, hPCSK9 with a point mutation ofD374Y (hPCSK9 (D374Y)-mmh) was generated.

Binding specificity of mAbs to test proteins hPCSK9-mmh, mousePCSK9-mmh, chimeric proteins #1, #2, and #3, and hPCSK9 (D374Y)-mmh weretested as follows: the mAbs were coated on a 96-well plate overnight at4° C., then each test protein (1.2 nM) was added to the plate. After 1hr binding at RT, the plate was washed and bound test protein detectedusing HRP-conjugated anti-myc polyclonal antibody (++=OD>1.0; +=OD0.4-1.0; −=OD<0.4).

TABLE 19 Chimeric Protein Antibody hPCSK9 mPCSK9 #1 #2 #3 hPCSK9(D374Y)H1M300 ++ − ++ + − ++ H1M309 ++ − − − ++ ++ H1M312 ++ − − − ++ ++ H1M492++ − − − − + H1M493 ++ − − − ++ ++ H1M494 ++ − − + ++ ++ H1M495 ++ − − −++ ++ H1M496 ++ − − − ++ ++ H1M497 ++ − − ++ + ++ H1M498 ++ − − − + ++H1M499 ++ − − − ++ ++ H1M500 ++ − ++ − − ++ H1M502 ++ − − − ++ ++ H1M503++ − − ++ − ++ H1M504 ++ − − − − + H1M505 ++ − ++ + − ++ H1M508 ++ − − −++ ++ H1H318P ++ − ++ − − ++ H1H334P ++ − ++ − − ++ H1H316P ++ ++ ++ ++++ ++ H1H320P ++ − − ++ − ++ Control I ++ − − − ++ ++

Binding specificity of 316P, 300N and control anti-hPCSK9 mAbs tohPCSK9-mmh, mPCSK9-mmh, mmPCSK9-mmh, mfPCSK9-mmh, rPCSK9-mmh, chimericproteins #1, #2, and #3, and hPCSK9 (D374Y)-mmh were tested as describedabove except that the protein concentration is 1.7 nM (−=OD<0.7; +=OD0.7-1.5; ++OD>1.5).

TABLE 20 316P 300N Control I Control II Control III hPCSK9-mmh ++ ++ ++++ ++ mPCSK9-mmh ++ − − ++ ++ mmPCSK9-mmh ++ ++ ++ ++ ++ mfPCSK9-mmh ++++ ++ ++ ++ rPCSK9-mmh ++ − − ++ + Chimeric Protein #1 ++ ++ − ++ ++Chimeric Protein #2 ++ ++ − ++ ++ Chimeric Protein #3 ++ + ++ ++ ++hPCSK9 (D374Y) ++ ++ ++ ++ ++

Similar results for selected mAbs were obtained by BIACORE™ bindingassay, Briefly, 316P, 300N, or Control I mAb was captured on anamine-coupled anti-hFc CM5 chip and 100 nM of each protein injected overthe mAb-captured surface. Changes in RU due to the binding of eachprotein to the mAb surface was determined.

TABLE 21 Chimeric Protein Antibody hPCSK9 mPCSK9 #1 #2 #3 316P 500 505529 451 467 300N 320 13 243 76 10 Control I 65 7 4 3 69

To further assess the binding specificity of 316P, which cross-reactswith mPCSK9-mmh, a cross-competition ELISA assay was developed todetermine binding domain specificity. Briefly, mAbs specific forchimeric protein #1, #2, or #3, were first coated on a 96-well plateovernight at 1 μg/ml. Human PCSK9-mmh (2 μg/ml) was then added to eachwell followed by 1 hr incubation at RT. 316P (1 μg/ml) was added andincubated for another hour at RT. Plate-bound 316P was detected usingHRP-conjugated anti-hFc polyclonal antibody. Although 316P binding tohPCSK9-mmh was not affected by the presence of mAbs specific for eitherchimeric protein #2 or chimeric protein #3, 316P binding to hPCSK9-mmhwas greatly reduced by the presence of antibody specific for chimericprotein #1.

Example 10 BIACORE™-Based Antigen Binding Profile Assessment

Antibody binding profiles were also established for 316P, 300N, ControlI, U, and In mAbs using BIACORE™ 1000. Briefly, hPCSK9-mmh was capturedon an anti-myc surface. A first anti-hPCSK9 mAb (50 μg/ml) was injectedover the PCSK9-bound surface for 10 min, at a flow rate of 10 μl/min at25° C. A second anti-hPCSK9 mAb (50 μg/ml) was then injected over thefirst mAb-bound surface for 10 min, at a flow rate of 10 μl/min at 25°C. Ability of the first mAb to block binding of the second mAb wasmeasured and is expressed as percent inhibition.

TABLE 22 Second mAb First mAb 316P 300N Control I Control II Control III316P 100 101 27 99 101 300N 77 100 12 82 −2 Control I 6 12 100 6 9Control II 91 102 −6 100 3 Control III 73 10 −12 1 100

Example 11 Increase of LDL Uptake by Anti-hPCSK9 Antibodies

The ability of anti-hPCSK9 mAbs to increase LDL uptake in vitro wasdetermined using a human hepatocellular liver carcinoma cell line(HepG2). HepG2 cells were seeded onto 96-well plates at 9×10⁴ cells/wellin DMEM complete media and incubated at 37° C., 5% CO₂, for 6 hr to formHepG2 monolayers. Human PCSK9-mmh, at 50 nM in lipoprotein deficientmedium (LPDS), and a test mAb was added in various concentrations from500 nM to 098 nM in LPDS medium. Data are expressed as IC₅₀ values foreach experiment (IC₅₀=antibody concentration at which increases LDLuptake by 50%). In addition, the experiment also showed that both 316Pand 300N were able to completely reverse the inhibitory effect of hPCSK9on LDL uptake, while Control I mAb or H1M508 anti-hPCSK9 mAb reversedthe inhibitory effect by about 50%.

TABLE 23 Antibody IC₅₀ (nM) 316P 21.30 300N 22.12 Control I >250 H1M508>250

The ability of anti-hPCSK9 mAbs to reverse the inhibitory effect on LDLuptake by PCSK9 protein from different mammalian species was also testedin a HepG2 cell line as described above. Briefly. HepG2 cells wereincubated overnight with serial dilutions of antibody in LPDS medium(beginning with 500 nM) and 50 nM of hPCSK9-mmh, mfPCSK9-mmh,mPCSK9-mmh, rPCSK9-mmh, or maPCSK9-h. HepG2 cells were also incubatedovernight with serial dilutions of antibody in LPDS (beginning with 50nM) and 1 nM hPCSK9(D374Y). As shown in Table 24, while 316P was able tocompletely reverse the inhibitory effect on LDL by all PCSK9 proteinstested, 300N was only able to reverse the inhibitory effect on LDLuptake by hPCSK9, hPCSK9 (D374Y), and mfPCSK9. Values are expressed asnM IC₅₀.

TABLE 24 316P 300N Control I Control II Control III hPCSK9-mmh 14.112.6 >500 13.4 12.4 hPCSK9(D374Y)-mmh 2.1 11 >50 0.7 0.6 mfPCSK9-mmh14.7 13.4 >500 14.2 13.6 mPCSK9-mmh 21.2 >500 >500 19 >500 rPCSK9-mmh27.7 >500 >500 21.9 >500 maPCSK9-h 14.4 >500 >500 29.5 127

Example 12 Neutralization of Biological Effect of hPCSK91n Vivo

To assess the biological effect of neutralizing PCSK9, hPCSK9 wasover-expressed in C578L/6 mice by hydrodynamic delivery (HDD) of DNAconstructs encoding full-length hPCSK9-mmh. 4 mice (C57BL/6) wereinjected with empty vector/saline (control), and 16 mice were injectedwith a 50 μg hPCSK9-mmh-DNA/saline mixture in the tail vein equal to 10%of their body weight. At day 7 after HDD, delivery of hPCSK9 resulted ina 1.6-fold elevation of total cholesterol, 3.4-fold elevation inLDL-cholesterol (LDL-C) and a 1.9-fold elevation in non-HDL cholesterol(relative to control). Serum hPCSK9 levels on day 7 were all greaterthan 1 μg/ml, as assessed by quantitative ELISA.

Administration of H1M300N on day 6 after HDD to 3 experimental groups(1, 5 or 10 mg/kg) (n=4 per group) via intraperitoneal (i.p.) injectionresulted in a significant attenuation of serum cholesterol levels. At 18hours after administration, total cholesterol was reduced by 9.8%, 26.3%and 26.8%, LDL-C was reduced by 5.1%, 52.3% and 56.7%, and non-HDLcholesterol was reduced by 7.4%, 33.8% and 28.6% in the 1, 5 or 10 mg/kgH1M300N treated groups, respectively.

Example 13 Pharmacokinetic and Serum Chemistry Study in Monkeys

A pharmacokinetic (PK) study was conducted in naïve male cynomolgusmonkeys (Macaca fascicularis) with a body weight range between 5-7 kgand aged between 3-5 years.

Group Assignments.

The monkeys were assigned into 5 treatment groups: Treatment Group 1(n=3) received control buffer (10 mM sodium phosphate, pH 6, 1 ml/kg);Treatment Group 2 (n=3) received 1 ml/kg of 316P (5 mg/ml); TreatmentGroup 3 (n=3) received 1 ml/kg 300N (5 mg/ml); Treatment Group 4 (n=3)received 1 ml/kg 316P (15 mg/ml); and Treatment Group 5 (n=3) received 1ml/kg 300N (15 mg/ml). All treatments were administered by IV bolusfollowed by a 1 ml saline flush. Total dose volume (ml) was calculatedon the most recent body weight (each animal was weighed twice duringacclimation and once weekly throughout the study). A single dose of testmAb or buffer control was administered on Day 1.

Animal Care.

Animals were housed in a temperature- and humidity-monitoredenvironment. The targeted range of temperature and relative humidity wasbetween 18-29° C. and 30-70%, respectively. An automatic lighting systemprovided a 12-hour diurnal cycle. The dark cycle could be interruptedfor study- or facility-related activities. The animals were individuallyhoused in cages that comply with the Animal Welfare Act andrecommendations set forth in The Guide for the Care and Use ofLaboratory Animals (National Research Council 1996).

Diet and Feeding.

Animals were fed twice per day according to SNBL USA SOPs. Animals werefasted when required by specific procedures (e.g., prior to blood drawsfor serum chemistry, urine collection, or when procedures involvingsedation are performed). The diet was routinely analyzed forcontaminants and found to be within manufacturer's specifications. Nocontaminants were expected to be present at levels that would interferewith the outcome of the study.

Experimental Design.

An appropriate number of animals were selected from SNBL USA stock.Animals were examined for health by veterinary staff, and had undergoneserum chemistry, hematology, and coagulation screening. Sixteen males,confirmed healthy, were assigned to the study. Fifteen males wereassigned to specific study groups and the remaining animal was availableas a spare. A stratified randomization scheme incorporating serumcholesterol level (based on the average of two draws in acclimation) wasused to assign animals to study groups.

Acclimation Period.

Previously quarantined animals were acclimated to the study room for aminimum of 14 days prior to initiation of dosing. Acclimation phase datawas collected from all animals, including the spare. All animals wereassessed for behavioral abnormalities that could affect performance onstudy. The spare animal was returned to stock after day 1.

Blood Collection.

Blood was collected by venipuncture from a peripheral vein fromrestrained, conscious animals. Whenever possible, blood was collectedvia a single draw and then divided appropriately.

PK Study.

Blood samples (1.5 ml) were collected at pre-dose, 2 min, 15, min, 30min, 1 hr, 2 hr, 4 hr, 8 hr, 12 hr, 24 hr, and subsequently once every24 hr in serum separator tubes (SST). Specimen storage serum istransferred to 2 vials and stored at −60° C. or below.

Serum samples were analyzed using an optimized ELISA (enzyme-linkedimmunosorbant assay) procedure. Briefly, a microtiter plate was firstcoated with hPCSK9-mmh. Test mAb 316P or 300N was then captured on thehPCSK9-mmh plate. The captured 316P or 300N was detected using abiotinylated mouse anti-hIgG4 followed by binding to NeutrAvidin-HRP.Varying concentrations of 316P or 300N, ranging from 100 to 1.56 ng/ml,were used as standards. One percent monkey serum (assay matrix) in theabsence of 316P or 300N was used as the zero (0 ng/ml) standard. Theresults, shown in FIG. 2, indicate a dose-dependent increase in serum316P and 300N levels. PK parameters were analyzed using WinNonlinsoftware (Noncompartmental analysis, Model 201—IV bolus administration).

TABLE 25 316P 300N PK Parameter 5 mg/kg 15 mg/kg 5 mg/kg 15 mg/kgT_(max) (h) 0.428 0.105 4.02 0.428 C_(max) (μg/ml) 184 527 226 1223T_(1/2) (h) 83 184 215 366

Serum Chemistry.

Blood samples were collected at pre-dose, 12 hr, 48 hr, and subsequentlyonce every 48 hr, for clinical chemistry analysis, in particular lipidprofiles (i.e. cholesterol, LDL-C, HDL-C, triglycerides). With theexception of the 12 hr post-dose sample, all animals were subject to anovernight fast prior to sample collection. The sample volume wasapproximately 1 ml. Chemistry parameters were determined using anOlympus automated analyzer. Parameters measured (Xybion code): Albumin(ALB); Alkaline Phosphatase (ALP); Alanine Aminotransferase (ALT);Aspartate Transaminase (AST); Total Bilirubin (TBIL); Calcium (Ca);Total Cholesterol (TCho); Creatine Kinase (CK); Creatinine (CRN); GammaGlutamyltransaminase (GGT); Glucose (GLU); Inorganic Phosphorus (IP);Total Protein (TP); Triglyceride (TRIG); Blood Urea Nitrogen (BUN);Globulin (GLOB); Albumin/Globulin Ratio (A/G); Chloride (Cl); Potassium(K); Sodium (Na); LDL and HDL cholesterol. Residual serum was stored at−20° C. or below and disposed of no sooner than one week after analysis.

Results from samples through Day 105 post-dose time point are shown inFIGS. 3-7. There was a reduction in total cholesterol and LDL-C inanimals receiving 316P and 300N, regardless of dose, within 24 hours ofthe first dose. Serum total cholesterol reduced rapidly and robustly(˜35%, FIG. 3). A robust decrease of ˜80% was seen in LDL-C (FIGS. 4-5)by day 6. In animals that received a 15 mg/kg dose of 300N, thereduction in both total cholesterol (˜10-15% reduction) and LDL-C (˜40%reduction) continued to at least day 80 of the study. In addition, HDL-Cwas elevated in animals that received 316P at 15 mg/kg (FIG. 6). Animalsthat received a higher dose (15 mg/kg) of either 316P or 300N alsoshowed a reduction in triglycerides during the course of study (FIG. 7).316P exhibited maximal suppression of LDL-C levels of up to 80% relativeto baseline. The length of this suppression was dose-dependent with atleast 60% suppression (relative to baseline LDL-C levels) lastingapproximately 18 days (5 mg/kg dose) and approximately 45 days (15 mg/kgdose). 300N exhibits a distinct pharmacodynamic profile from 316P. LDL-Csuppression by 300N was sustained for a much longer period of time atcomparable doses (50% LDL-C suppression for 28 days following a 5 mg/kgdose and 50% LDL-C suppression for approximately 90 days following a 15mg/kg dose). There was little or no measurable change in liver functionas determined by ALT and AST measurements. All animals receiving ananti-PCSK9 antibody in the study exhibited a rapid suppression If LDL-Cand total cholesterol.

A similar LDL-C lowering effect of 316P and 300N was also observed incynomolgous monkeys that received a single subcutaneous (SC)administration of either 5 mg/kg 316P or 5 mg/kg 300N (FIG. 8). Both316P and 300N dramatically suppressed LDL-C levels and maintained anLDL-C lowering effect for approximately 15 and 30 days, respectively(FIG. 8). The pharmacodynamic effect (approximately 40% LDL-Csuppression) approximately correlates with functional antibody levels inmonkey serum (FIG. 9). As antibody levels decrease below 10 μg/ml, LDL-Csuppression appeared to diminish as well. In addition, 300N demonstrateda substantially longer circulating half-life than 316P and hence alonger observed LDL-C suppression.

TABLE 26 PK Parameter 316P 300N T_(max) (h) 60 84 C_(max) (μg/ml) 46 63T_(1/2) (h) 64 286

Example 14 Attenuation of LDL Receptor Degradation by Anti-hPCSK9Antibodies

To assess the biological effect of PCSK9 on hepatic LDL receptor levelsand subsequent effects on serum LDL-C levels, hPCSK9 was administered tomice expressing hPCSK9 but not mPCSK9 (PCSK9^(hu/hu) mice) byintravenous injection. Specifically, PCSK9^(hu/hu) mice were injectedwith PBS (control), or 1.2 mg/kg hPCSK9-mmh via the tail vein. Six hoursafter delivery of hPCSK9, a 1.4-fold elevation (relative to baselinelevel) in total cholesterol and a 2.3-fold elevation in LDL-C) in serumwere observed. Analysis of hepatic LDL receptor levels in a separatecohort (n=3) of animals 4 hours after hPCSK9 administration revealed asignificant reduction in detectable LDL receptor in liver homogenates.

To assess the biological effect of anti-hPCSK9 on hepatic LDL receptorlevels and subsequent effects on serum LDL-C levels, 316P and anon-hPCSK9 specific mAb were administered to PCSK9^(hu/hu) mice atequivalent dose (5 mg/kg i.p.) 20 hours prior to the hPCSK9-mmh proteininjection described above. Four hours after the hPCSK9 administration,mice were sacrificed and a total of eight tissues (liver, brain, lung,kidney, heart, ileum, adrenal, and pancreas) were collected and levelsof LDL receptor were determined by Western blot. Changes in LDL receptorlevels were only observed in liver. In comparison to PBS control dosing,administration of 316P significantly blocked the PCSK9-mediatedincreases in total cholesterol and LDL cholesterol (LDL-C=2.49 mg/dl atbaseline and 3.1 mg/dl 6 hours after PCSK9; a 25% increase compared to135% with vehicle). Prior administration of the non-hPCSK9 specific mAbblocked LDL-C increases by approximately 27% from PBS alone (LDL-C=4.1mg/dl compared to PBS 5.6 mg/dl). Analysis of LDL receptor levels in aseparate cohort of mice (n=3 per treatment group) revealed a significantreduction in LDL receptor levels with PCSK9 administration, which wasblocked by 316P but not by the non-hPCSK9 specific mAb (FIG. 10).

Effect of different doses of 316P was also evaluated in PCSK9^(hu/hu)mice with both elevated LDL-C and elevated hPCSK9 levels. PCSK9^(hu/hu)mice were first placed on a high carbohydrate diet for 8 weeks,resulting in a ˜2-fold elevation in both LDL-C and hPCSK9 levels. Either316P or a non-hPCSK9 specific mAb, each at 1 mg/kg, 5 mg/kg, or 10mg/kg, were administered to the mice. Sera were collected 24 hours laterand LDL-C levels were analyzed. 316P was effective in decreasing LDL-Clevels in a dose-dependent manner (FIG. 11). In addition, 316Padministered at a dose of 10 mg/kg, rapidly reduced LDL-C levels back tooriginal (pre-diet) values within 24 hours.

Example 15 Mouse PK Studies

A PK study was conducted in 6-week-old C57BL/6 mice and 11-15 week oldhPCSK9 heterozygous mice. A single injection of Control I. 316P, or300N, each at 10 mg/kg, was administered SC. Serum bleeds were measuredfor hIgG levels at 0 hr (pre-bleed), 6 hr, day 1, 3, 6, 10, 14, 21, 28,35, 42 and 56, for a total of 12 time points, using an anti-hFc captureand anti-hFc detection sandwich ELISA (FIGS. 12 and 13). All mAbsachieved their T_(max) at approximately 3 days with correspondingC_(max) levels of approximately 47-115 μg/ml for C57BL/6 mice and 55-196μg/ml for hPCSK9 heterozygous mice. At. Day 56, Control I mAb levelswere about 12 μg/ml and 300N levels were about 11 μg/ml whereas 316Plevels were about less than 0.02 μg/ml in C57BU6 mice. At Day 56 inhPCSK9 heterozygous mice, Control I mAb levels were about 29 μg/ml,while both 300N and 316P levels were below the quantifiable limit (BOL)of 0.02 μg/ml.

Example 16 Anti-hPCSK9 Antibody Binding to Mutant/Variant hPCSK9

To further assess binding between hPCSK9 and anti-hPCSK9 mAbs, 21variant hPCSK9 proteins in which each variant contained a single pointmutation and two variant hPCSK9 proteins each contained a doublemutation were generated. Each selected antibody was captured on aF(ab′)2 anti-hIgG surface created through direct chemical coupling to aBIACORE™ chip to form a captured antibody surface. Each mmh-taggedvariant hPCSK9 at varying concentrations from 100 nM to 25 nM was theninjected over the captured antibody surface at a flowrate of 60 μl/minfor 240 sec, and the dissociation of variant hPCSK9 and antibody wasmonitored in real time for 20 min at 25° C. nb: no binding was observedunder these experimental conditions (K_(D)=M×10⁻⁹; T_(1/2)=min;WT=wildtype).

TABLE 27 316P 300N Control I Control II Control III K_(D) T_(1/2) K_(D)T_(1/2) K_(D) T_(1/2) K_(D) T_(1/2) K_(D) T_(1/2) WT 1.00 37 0.69 12030.6 16 0.10 333 0.60 481 P70A 1.42 32 1.68 80 19.0 16 0.24 168 0.90 325S127R 2.40 36 1.87 110 25.0 18 0.26 288 0.55 550 D129G 1.27 36 1.40 8822.9 18 0.19 257 0.75 445 S147F 1.29 32 9.07 24 21.1 15 0.22 178 0.231468 S153R 5.64 4 0.56 141 36.6 17 0.09 322 3.33 60 E159R 6.96 5 0.82 9431.7 16 0.08 350 2.97 68 T162R 0.98 43 0.58 140 29.0 17 0.09 322 0.48362 D192R 1.35 28 0.75 119 30.2 15 0.09 326 nb nb R194E 0.38 71 0.65 12931.4 16 0.07 389 nb nb E197R 1.42 27 0.67 115 30.2 17 0.09 339 nb nbR215H 0.86 41 1.03 98 37.8 17 0.65 49 0.74 272 R215E 0.90 43 1.81 7744.0 16 4.48 12 0.78 276 F216L 1.83 32 0.99 121 21.2 15 1.35 39 0.33 880R237E 2.48 15 1.03 109 29.6 15 0.07 481 5.89 43 D238R 410 1 0.78 12325.9 19 0.24 144 0.14 1273 A341R 1.54 21 0.34 190 28.7 18 0.08 340 0.88200 D343R 7.88 6 1.18 89 27.0 16 0.08 402 4.13 66 R357H 6.26 30 6.53 6626.4 13 0.63 165 1.91 896 E366K 2.92 13 36.0 2 28.8 18 0.46 69 0.38 808D374Y 2.04 15 0.66 83 25.0 17 0.08 285 1.02 161 V380M 0.48 63 2.82 2825.9 17 0.15 177 0.35 711 P70A, S147F 1.18 34 7.87 24 23.5 18 0.23 1640.79 348 E366K, V380M 3.33 12 78.3 1 25.5 18 0.59 60 0.52 551

The results show that when residue D238 was mutated, the bindingaffinity of 316P for hPCSK9 was reduced >400-fold, from a K_(D) of1×10⁻⁹ M to 410×10⁻⁹ M; and T_(1/2) shortened about 30-fold, from 37 to1 min, indicating that 316P binds an epitope on hPCSK9 comprising D238of hPCSK9 (SEQ ID NO:755). Additionally, BIACORE™ assays show that 316Pbinding affinity and T_(1/2) were reduced about 5- to 10-fold when aresidue at 153, 159 or 343 was mutated. Specifically, K_(D) was reducedfrom about 1×10⁻⁹ M to between about 5-8×10⁻⁹ M when any one of S153,E159 or D343 were mutated; while T_(1/2) was decreased from about 37 minto between about 4-6 min.

300N binding to hPCSK9 was reduced about 50-fold when the residue atposition 366 was mutated, resulting in a decreased K_(D) of from about0.7×10⁻⁹ M to about 36×10⁻⁹ M and a shorter T_(1/2) from about 120 to 2min. These results indicate that 300N binds an epitope on hPCSK9comprising E366 of hPCSK9 (SEQ ID NO:755). Additionally, the BIACORE™assays show that 300N binding affinity and T_(1/2) were reduced between2- to >10-fold when a residue at 147 or 380 was mutated. Specifically,K_(D) was reduced from about 0.69×10⁻⁹ M to between about 2-9×10⁻⁹ Mwhen any of S147 or V380 were mutated; while T_(1/2) was shortened fromabout 120 min to between about 24-66 min. Compared to 316P, 300N bindingto hPCSK9 was not reduced by a mutation at residue 238.

In contrast, Control I antibody did not exhibit an altered bindingaffinity or T_(1/2) in response to any of the positional mutationstested; Control II antibody exhibited a 40-fold decreased affinity whenresidue 215 was mutated (R215E) (from ˜0.1×10⁻⁹ to ˜4.5×10⁻⁹), andT_(1/2) was about 27-fold shorter (from ˜333 to 12 min); while ControlIII antibody exhibited a decreased affinity when residue 237 was mutated(K_(D) decreased from ˜0.6×10⁻⁹ to ˜5.9×10⁻⁹, and T_(1/2) decreased from˜481 to ˜43 min).

Binding specificity of 316P, 300N, and control anti-hPCSK9 mAbs tohPCSK9 variants was tested using an ELISA-based immunoassay. Anti-PCSK9mAbs were coated on a 96-well plate overnight at 4° C. Each mmh-taggedvariant hPCSK9 in CHO-k1 transient transfection lysate supernatants wasadded to the antibody-coated plate at various concentrations rangingfrom 0 to 5 nM. After 1 hr binding at RT, the plate was washed and boundvariant hPCSK9 was detected using HRP-conjugated anti-myc polyclonalantibody (−=OD<0.7; +=OD 0.7-1.5; ++=OD>1.5).

TABLE 28 Control Control Control hPCSK9 or Variant 316P 300N I II IIIhPCSK9(WT) ++ ++ ++ ++ ++ hPCSK9(S127R) ++ ++ ++ ++ ++ hPCSK9(D129G) ++++ ++ ++ ++ hPCSK9(S153R) ++ ++ ++ ++ ++ hPCSK9(R215H) ++ ++ ++ ++ ++hPCSK9(F216L) ++ ++ ++ ++ ++ hPCSK9(R237E) ++ ++ ++ ++ ++ hPCSK9(D238R)− ++ ++ ++ ++ hPCSK9(A341R) ++ ++ ++ ++ ++ hPCSK9(D343R) ++ ++ ++ ++ ++hPCSK9(R357H) ++ ++ ++ ++ ++ hPCSK9(E159R) ++ ++ ++ ++ ++ hPCSK9(T162R)++ ++ ++ ++ ++ HPCSK9(D192R) ++ ++ ++ ++ − hPCSK9(R194E) ++ ++ ++ ++ −hPCSK9(E197R) ++ ++ ++ ++ − hPCSK9(R215E) ++ ++ ++ ++ ++ hPCSK9(P70A) ++++ ++ ++ ++ hPCSK9(S147F) ++ ++ ++ ++ ++ hPCSK9(E366K) ++ + ++ ++ ++hPCSK9(V380M) ++ ++ ++ ++ ++ hPCSK9(P70A, S147F) ++ ++ ++ ++ ++hPCSK9(E366K, V380M) ++ + ++ ++ ++

Example 17 Effect of 316P on Normolipemic and Hyperlipemic Hamster

The ability of anti-PCSK9 mAb 316P to reduce serum LDL-C was tested innormolipemic or hyperlipemic Gold Syrian hamsters (Mesocricetusauratus). Male Syrian Hamsters, age 6-8 weeks, weighing between 80-100grams, were allowed to acclimate for a period of 7 days before entryinto the study. All animals were placed on either a standard chow dietor a hyperlipemic diet of chow supplemented with 0.1% cholesterol and10% coconut oil. The 316P mAb was delivered to hamsters by a singlesubcutaneous injection at doses of 1, 3, or 10 mg/kg for normolipemichamsters and at doses of 3, 10, or 30 mg/kg for hyperlipemic hamsters.Serum samples were taken from all groups at 24 hr and 7, 14, and 22 dayspost injection, at which time serum lipid levels were assessed andcompared to baseline levels taken 7 days prior to the administration ofthe mAbs. Circulating total cholesterol and LDL-C in normolipemichamsters was significantly reduced in a dose-dependent manner comparedto vehicle injection. As shown in FIG. 14, administration of 316Peffectively reduced LDL-C levels by up to 60% seven days post injectionat the highest dose (10 mg/kg) tested. Similar cholesterol reducingeffect of 316P was not observed in hyperlipemic hamsters.

Example 18 A Randomized, Double-Blind, Placebo-Controlled, AscendingSingle-to-Multi-Dose Study of the Safety, Tolerability, and Bioeffect ofSubcutaneously Administered Human Anti-PCSK9 Antibody in Patients withand without Concomitant Atorvastatin

The objective of this study was to determine whether a fully humanmonoclonal antibody to PCSK9 (mAb316P) is effective and safe as either aprimary or adjunctive agent to lower LDLc in patients with HeterozygousFamilial Hypercholesterolemia (HeFH) or other forms of primaryhypercholesteremia (nonFH).

This study was a randomized, double-blind, placebo-controlled, multipleascending dose clinical trial enrolling 61 adults with either documentedHeFH (n=21) or nonFH (n=30), on diet plus stable atorvastatin therapy(atorvaRx) or nonFH (n=10) on diet alone. Subjects on stableatorvastatin therapy had LDLc≧2.6 mmol/L and those on diet alone hadLDLc≧3.4 mmol/L. mAb316P at doses of 50, 100 and 150 mg was administeredsubcutaneously (sc) at 1, 29 and 43 days. The primary endpoint was theincidence and severity of treatment emergent adverse events (TEAS). Theprimary efficacy endpoint was percent and absolute change in serum LDLcfrom baseline to each visit. Additional endpoints includedapolipoprotein (apo) B, total cholesterol, HDLc, VLDLc, and the ratio ofapoB to apoA1.

109 patients were screened, and 61 patients were randomized (14 placebo,47 mAb316P) with 100% completing 148+/−7 days of treatment and followup. Compared to the nonFH cohort, the FH group was younger (mean 40 vs52 yrs), had more males (81% vs 57%) and was on higher doses ofatorvastatin (52% on 40 mg vs 3%). Baseline LDLc was 3.45, 2.88 and 4.46mmol/L respectively in the FH, nonFH atorvaRx and nonFH diet only groupsrespectively. Response to mAb316P (expressed as percent change incalculated serum LDL-C from baseline to each visit) is shown Tables 29and 30. Treatment with mAb316P resulted in mean % reductions in LDLC ontop of statins on day 57 of 35.6%, 50.2% and 57.5% at the 50, 100 and150 mg doses, respectively, in the combined FH and nonFH populations.There did not appear to be differences in response between FH and nonFHor those on or not on statin therapy.

Favourable changes were also observed in HDLC and apoA1. No seriousadverse events were seen and treatment was generally well tolerated. Nodrug-related adverse effects were seen on liver function testing orother laboratory parameters.

TABLE 29 Percent Change in Calculated Serum LDL-C From Baseline to EachVisit* FH Patients on Atorvastatin Non-FH Patients on Atorvastatin AbDose: PBO^(#) 50 mg 100 mg 150 mg PBO^(#) 50 mg 100 mg 150 mg patients:(N = 6) (N = 5) (N = 5) (N = 5) (N = 6) (N = 8) (N = 8) (N = 8) Baseline— — — — — — — — Day 1 Visit 5 2.03 −0.42 −7.50 −5.88 3.16 −8.98 −10.09−14.29 Day 2 (6.119) (4.603) (6.084) (10.366) (9.748) (5.819) (11.047)(8.751) Visit 6 0.39 −4.81 −20.91 −15.21 5.67 −21.28 −27.27 −21.00 Day 3(7.522) (7.306) (10.160) (11.538) (8.135) (5.678) (16.699) (14.256)Visit 7 −2.79 −30.44 −50.96 −40.81 −6.62 −43.13 −56.95 −46.81 Day 8 ± 3(5.318) (10.776) (16.227) (20.082) (8.384) (6.406) (19.049) (19.233)Visit 8 6.36 −31.42 −53.67 −52.95 4.48 −38.71 −54.36 −62.00 Day 15 ± 3(19.607) (18.218) (12.128) (17.130) (7.389) (11.028) (7.819) (16.531)Visit 9 10.20 −4.99 −21.07 −27.03 −0.63 −2.24 −11.48 −17.64 Day 29 ± 3(14.274) (9.479) (16.407) (21.567) (13.983) (16.704) (20.396) (14.132)Visit 10 1.86 −32.31 −47.59 −44.47 7.54 −30.88 −50.53 −55.72 Day 43 ± 3(14.283) (15.685) (13.104) (27.321) (10.473) (13.053) (10.389) (11.393)Visit 11 3.45 −39.26 −53.64 −55.80 5.84 −33.36 −48.04 −58.52 Day 57 ± 3(9.693) (8.294) (12.404) (15.596) (14.883) (8.700) (9.366) (17.918)Visit 12 2.30 −9.02 −19.17 −23.24 3.54 −2.77 −13.80 −15.84 Day 71 ± 3(18.929) (7.955) (16.643) (29.233) (17.026) (11.065) (25.640) (13.593)Visit 13 −1.70 −2.72 −7.04 −9.82 10.90 −2.01 9.18 6.66 Day 85 ± 3(14.163) (16.512) (15.835) (21.450) (23.826) (10.720) (28.556) (14.575)Visit 14 4.93 5.76 −1.76 4.32 4.62 1.23 −2.53 14.77 Day 99 ± 3 (18.181)(10.957) (9.717) (20.651) (16.912) (16.703) (13.430) (16.167) Visit 150.21 2.29 0.36 8.26 −0.92 1.76 0.89 13.06 Day 120 ± 3 (17.738) (7.043)(14.954) (50.237) (23.154) (12.863) (13.837) (16.902) Visit 16 4.67−3.02 5.32 4.23 1.79 5.40 8.63 12.43 Day 148 ± 3 (18.920) (6.420)(21.592) (35.706) (28.237) (17.012) (21.463) (19.139) ^(#)PBO = placebo*Values represent Mean percent change from baseline (Standard Deviation)

TABLE 30 Percent Change in Calculated Serum LDL-C From Baseline to EachVisit* FH and Non-FH Patients on Non-FH Patients Not on Atorvastatin,Combined Atorvastatin Ab Dose: PBO^(#) 50 mg 100 mg 150 mg PBO^(#) 150mg patients: (N = 12) (N = 13) (N = 13) (N = 13) (N = 2) (N = 8)Baseline — — — — — — Day 1 Visit 5 2.60 −5.69 −9.10 −11.05 7.13 −7.28Day 2 (7.782) (6.753) (9.233) (9.930) (2.911) (4.156) Visit 6 3.03−14.94 −24.82 −18.78 11.64 −11.37 Day 3 (7.963) (10.302) (14.403)(13.097) (2.323) (7.661) Visit 7 −4.70 −38.25 −54.65 −44.50 12.52 −41.59Day 8 ± 3 (6.985) (10.193) (17.567) (18.959) (13.260) (13.106) Visit 85.42 −35.90 −54.09 −58.52 2.75 −44.68 Day 15 ± 3 (14.161) (13.971)(9.209) (16.681) (17.896) (15.461) Visit 9 4.79 −3.30 −15.17 −21.2515.09 −38.57 Day 29 ± 3 (14.610) (13.952) (18.867) (17.152) (20.319)(14.306) Visit 10 4.70 −31.43 −49.40 −51.39 4.71 −50.88 Day 43 ± 3(12.304) (13.488) (11.064) (18.893) (6.661) (11.674) Visit 11 4.64−35.63 −50.19 −57.47 6.09 −54.41 Day 57 ± 3 (12.040) (8.717) (10.513)(16.439) (28.082) (12.175) Visit 12 2.92 −5.17 −15.86 −18.68 16.05−42.16 Day 71 ± 3 (17.177) (10.126) (21.982) (20.167) (25.084) (29.771)Visit 13 4.60 −2.28 2.94 0.32 14.58 −30.13 Day 85 ± 3 (19.813) (12.572)(25.034) (18.627) (7.290) (21.347) Visit 14 4.78 2.98 −2.23 10.75 7.50−11.83 Day 99 ± 3 (16.742) (14.423) (11.698) (17.963) (12.321) (18.493)Visit 15 −0.35 1.97 0.68 11.21 25.69 −8.36 Day 120 ± 3 (19.674) (10.636)(13.649) (31.840) (14.125) (7.430) Visit 16 3.23 2.16 7.35 9.28 −6.29−0.74 Day 148 ± 3 (22.965) (14.168) (20.662) (25.611) (15.014) (13.169)^(#)PBO = placebo *Values represent Mean percent change from baseline(Standard Deviation)

It can be concluded from this study that mAb316P is an effectivetherapeutic option for patients with heFH or non-FH, with elevatedcholesterol, on statin therapy or on diet alone.

Example 19 Therapeutic Dose Determination of an Anti-PCSK9 Antibody toTreat Hyperchoiesterolemia Background

Patient treatment guidelines for dyslipidemia strive to reduce lowdensity lipoprotein cholesterol (LDL-C) levels to goals of ≦100 mg/dL or≦70 mg/dL depending on the level of cardiovascular disease risk. Mostpatients will require ˜50% reduction in LDL-C to reach those goals.Despite dramatic reductions in heart disease associated with existingstandard-of-care (including statins), substantial proportions ofhigh-risk patients do not attain LDL-C goals. In phase 2 studies of amonoclonal antibody to proprotein convertase subtilisin/kexin type 9,(mAb316P) 150 mg delivered via single 1 mL subcutaneous injection every2 weeks (Q2W) demonstrated LDL-C reductions (Least Squares means)independent of baseline levels ranging from 66.2% to 72.4%. The variousdosing regimens investigated exhibited a range of LDL-C reductions.Therefore, in this Example, a dose-response modeling analysis wasperformed to estimate a dose which would lead to ˜50% reduction inLDL-C.

Methods

Dose response modeling was performed using a Multiple ComparisonProcedures-Modeling approach on Q2W (i.e., once every two week) dosesfrom a double-blind, parallel-group, placebo-controlled, multicenter,dose-ranging clinical trial in patients with LDL-C≧100 mg/dL on stablestatin therapy. Three types of candidate dose-response models (linear,umbrella, logistic with 4 model shapes) were fitted to the data.

Results

Based on the selected logistic model, the 75 mg dose is expected toprovide a difference vs placebo in percent LDL-C change from baseline of−49.2% with a 95% confidence interval of [−57.4%; −40.9%]. In priorphase 2 studies (N=274 patients, doses ranging from 50 to 300 mg), themost common adverse events (AEs) were mild injection site reactions withno observed liver or muscle cell toxicity; 7 serious AEs occurred: 4treatment-related, 1 non-treatment-related, 2 in the placebo group.

CONCLUSION

Phase 3 studies of mAb316P in a range of hypercholesterolemicpopulations will include evaluation of a starting dose of 75 mg Q2W toprovide ˜50% reduction in LDL-C, with flexibility for up-titration to150 mg Q2W for patients who require higher doses to achieve LDL-C goals.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

1. A method for treating hypercholesterolemia in a subject, the methodcomprising sequentially administering to the patient a single initialdose of an antibody or antigen-binding fragment thereof whichspecifically binds hPCSK9, followed by one or more secondary doses ofthe antibody or antigen-binding fragment thereof; wherein each secondarydose is administered to the subject 1 to 4 weeks after the immediatelypreceding dose; and wherein the antibody or antigen-binding fragmentcomprises the heavy and light chain CDRs of a HCVR/LCVR amino acidsequence pair selected from the group consisting of SEQ ID NOs:90/92 and218/226.
 2. The method of claim 1, wherein the antibody orantigen-binding fragment comprises heavy and light chain CDR amino acidsequences having SEQ ID NOs:220, 222, 224, 228, 230 and
 232. 3. Themethod of claim 2, wherein the antibody or antigen-binding fragmentcomprises an HCVR having the amino acid sequence of SEQ ID NO:218 and anLCVR having the amino acid sequence of SEQ ID NO:226.
 4. The method ofclaim 1, wherein the antibody or antigen-binding fragment comprisesheavy and light chain CDR amino acid sequences having SEQ ID NOs:76, 78,80, 84, 86 and
 88. 5. The method of claim 4, wherein the antibody orantigen-binding fragment comprises an HCVR having the amino acidsequence of SEQ ID NO:90 and an LCVR having the amino acid sequence ofSEQ ID NO:92.
 6. The method of claim 1, wherein each secondary dose isadministered 2 weeks after the immediately preceding dose (Q2W).
 7. Themethod of claim 6, wherein the initial dose and the secondary doses eachcomprise between 25 mg to 200 mg of the antibody or antigen-bindingfragment thereof.
 8. The method of claim 7, wherein the initial dose andthe secondary doses each comprise 59 mg of the antibody orantigen-binding fragment thereof.
 9. The method of claim 7, wherein theinitial dose and the secondary doses each comprise 75 mg of the antibodyor antigen-binding fragment thereof.
 10. The method of claim 7, whereinthe initial dose and the secondary doses each comprise 100 mg of theantibody or antigen-binding fragment thereof.
 11. The method of claim10, wherein the initial dose and the secondary doses each comprise 150mg of the antibody or antigen-binding fragment thereof.
 12. The methodof claim 1, wherein the subject is on a therapeutic statin regimen atthe time of or just prior to administration of the initial dose of theantibody or antigen-binding fragment thereof.
 13. The method of claim12, wherein the therapeutic statin regimen comprises a statin selectedfrom the group consisting of cerivastatin, atorvastatin, simvastatin,pitavastatin, rosuvastatin, fluvastatin, lovastatin and pravastatin. 14.The method of claim 13, wherein the statin is atorvastatin.
 15. Themethod of claim 1, wherein the subject has heterozygous FamilialHypercholesterolemia (heFH).
 16. The method of claim 1, wherein thesubject has a form of hypercholesterolemia that is not FamilialHypercholesterolemia (nonFH).